In-vitro response of potato (Solanum tuberosum var. Rio Grand) to various sucrose and agar concentrations.
استجابة نبات البطاطس صنف ريو جراند لتركيزات مختلفة من السكروز والأجار.
Dow M مفتاح محمد ضو Faculty of Agriculture, University of Tripoliكلية الزراعة/ جامعة طراابلس
Shwerif Mملاك محمد الشويرف Faculty of Agriculture, University of Tripoliكلية الزراعة/ جامعة طراابلس
Barbaraui Aاحمد محمد البربراوي Faculty of Agriculture, University of Tripoliكلية الزراعة/ جامعة طراابلس
Ben Hmaida Aعبد السلام بن احميدة Ministry of agriculture (Tripoli- Libya).وزارة الزراعة/ ليبيا
Abstract
Effects of sucrose and agar concentration on potato (Solanum tuberosum L) var. Rio Grand grown in vitro were investigated with three sucrose or agar levels (1, 2, and 3% w/v), (0.5, 0.6, and 0.7% w/v) respectively by using single node explants. Significant differences affects were noticed among the plantlets cultivated in different sucrose levels. Decreasing sucrose level less than the normal concentration (3%) of MS medium showed gradual decrease in all parameters tested including shoot length, leaflets number, shoot fresh mass, shoot dry mass, root number, root fresh mass, root dry mass; in contrast, root length was increased as sucrose level reduced in the medium.
Reducing agar concentration to 0.5% favored shoot and root fresh and dry weight; while, shoot length and leaflets number were not affected by agar level in the medium.
Keywords: Potato, in vitro, sucrose, agar, single node.
المستخلص:
صنف ريو جراند(Solanum tuberosum L.) على البطاطس تم دراسة تأثيرات تركيز السكروز و الأجار النامية باستخدام زراعة الأنسجة النباتية وذلك باستخدام 3 مستويات من كلاً من السكروز (1، 2، 3% وزن/حجم) والأجار (0.5، 0.6، 0.7% وزن/حجم) وباستخدام العقد النباتية المفردة. لوحظ وجود تأثيرات وبفروق معنوية بالنباتات التي زرعت على مستويات مختلفة من السكروز. أظهر مستوى السكروز اقل من المستوى العادي (3%) المستخدم في وسط نمو موراشيجي و سكوج (SM) أظهرت انخفاض تدريجي في كل الاختبارات المدروسة والتي تشمل طول الأفرخ الخضرية، عدد الأوراق، الوزن الطازج للأفرخ الخضرية، الوزن الجاف للأفرخ الخضرية، عدد الجذور، الوزن الطازج للمجموع الجذري، الوزن الجاف للمجموع الجذري، بالمقابل فأن طول الجذر ازداد كلما زاد مستوى السكروز في وسط النمو.
إن تقليل تركيز الأجار إلى 0.5% دعم الوزن الطازج والجاف لكلاً من المجموع الخضري والجذري، بينما طول الأفرخ الخضرية وعدد الوريقات لم يتأثران بمستوى الأجار في وسط النمو.
Introduction:
Potato is the most important food crop in the world in terms of its production, which only exceeded by those of rice, wheat, and maize. The estimated world production of potato in 2014 was as much as 320 million ton (5). The average yield of potato is considerably low because of a series of biotic and abiotic stresses. Of the biotic stresses, several viral and other diseases are the main factors whereas regarding abiotic stresses such as drought, salinity and heat are the main constraints of high yield (1). Plant breeders around the world are trying to develop high yielding and stress tolerant genotypes. For these purposes both conventional and non-conventional approaches are applied. Of the non-conventional techniques, in vitro or tissue culture approach is widely applied to improve potato production by means of micro-propagation in order to produce large number of plants in a very short time, to speed up the production and supply of new varieties into the market, and to maintain disease free stock of seed potatoes for further multiplication.
Since the beginning of in vitro culture in 1902 when the Austrian botanist Gottlieb Haberlandt attempted to grow isolated plant cells and tissues in nutritive solutions, a large body of work has emerged describing the optimization of different culture conditions to supply explants with all the components required for successful in vitro plant tissue propagation. During the past 80 years, more than 3000 scientific articles have described the use of over 2000 different culture media in plant tissue culture (10). In vitro tissue propagation, however; is still a stressful procedure for plants, which can limit the successful establishment of plants upon transfer to ex vitro conditions (2, 12). The main components of most plant tissue culture media are mineral salts and sugar as carbon source and water (8). Sugar in culture medium has been considered the sole carbon source for the growth of cells, buds, shoots, and even plantlets. Sugars enter the metabolic pathways and transformation of energy which are required for growth of cell (9). In plant tissue culture, photosynthesis is insufficient, due to the growth taking place in conditions unsuitable for photosynthesis or without photosynthesis (20). Impact of different carbohydrates with other constituents of nutrient media are reported in several studies (3, 25). The sugar concentration chosen is very dependent on the type and age of growth material; very young embryos require a relatively high sugar concentration. Generally, the growth and development increases with increased sugar concentration, until an optimum is reached and then decreases at higher concentrations. Among the sugars, sucrose is used as a principal carbon source for in vitro plant culture probably, because it is the most common carbohydrate in the phloem sap of many plants (6). Sucrose also supports the maintenance of osmotic potential and the conservation of water in cells. However, high sucrose concentration in the media restricts the photosynthetic efficiency of cultured plants by reducing the levels of chlorophyll, key enzymes for photosynthesis and epicuticular waxes promoting the formation of structurally and physiologically abnormal stomata (13). On the other hand, some studies have shown that plantlets growing under tissue culture conditions do not fix enough CO2 to sustain growth in the absence of sucrose, which is mainly due to limited CO2 inside the vessel (15).
Interaction between in vitro raised plantlets with the gelling agent in culture medium is a dynamic process and the changes in gel consistency affect the regeneration of plants or tissues (23). Traditionally, agar is added to the medium to increase its viscosity. Increasing agar strength beyond a critical limit has been demonstrated to inhibit organogenesis and shoot growth and reduce the water availability to the culture (24). Some reports have suggested that low concentration of agar provides a poorly gelled medium that facilitates adequate contact between the plant tissue and the medium and betted diffusion of medium constituents, resulting in better growth and their subsequent rooting (4).
The objectives of this study were to investigate the influence of sucrose and agar concentration in the medium on in vitro growth traits of potato (Solanum tuberosum var. Rio Grand) by using single-node plantlets.
Materials and methods:
This experiment was carried out in Agricultural Research Center-Ministry of agriculture, Tripoli-Libya. Potato tubers were obtained from a recognized company for importing seeds and tubers. Tubers were well washed and put in dark for few weeks until sprouts reaches 1-2 cm in length. Sprouts were surface sterilized with 70% ethanol for 1 min followed by 2% sodium hypochlorite for 5 min. three times, sprouts then washed 3 times by distilled water. Sterilized sprouts 1-2 cm length were cultured to obtain shoot culture in Murashige and Skoog (MS; 1962) medium having 3% sucrose, 0.7% Difco Bacto- agar, the medium was adjusted to pH 5.7 prior to autoclaving at 120 0C for 20 min. Cultures were maintained at 25±2 0C with 16/8 h D/N at 40 µmol m-2 s-1 photo flux density (cool white fluorescent light). After six weeks plantlets were cut into 1 cm-long single-node with 1 leaf and an axillary bud, two segments were implanted in each culture vessel (200 mL) containing 20 mL of MS medium. In order to study the effect of either different sucrose or agar (Difco Bacto) concentration on plantlets growth and development, the medium was modified using different sucrose (1, 2, and 3% w/v) or agar (0.5, 0.6, and 0.7% w/v). The experiment ended after 4 weeks and the growth response were noted on shoot length (SL), leaflets number (LN), shoot fresh mass (SF), shoot dry mass (SD), root length (RL), root number (RN), root fresh mass (RF), and root dry mass (RD). The experimental design used was a completely randomized design, twenty replicates per treatment were used, and every replicate contained two plantlets. Mean separation was analyzed by Duncan multiple range test at 5% level of significance.
Results and discussion
The MS-sucrose concentration and medium consistency directly influenced in vitro potato plantlets growth, sucrose and agar are important factors for plantlets growth. Different concentrations of sucrose in MS medium were used for growth of single node explants of potato plantlets. All tested traits were found to be effected by sucrose concentration. Decreasing sucrose level in the medium less than the normal concentration (3%) of MS medium showed gradual decrease in all parameters tested except root length which was increased respectively by 24%, and 39% at 2%, and 1% sucrose concentration in the medium (table 1). The percentage reduction of shoot length, leaflets number, shoot fresh mass, shoot dry mass, respectively, were 39%, 26%, 36%, and 75% at 2% sucrose and 52%, 27%, 53%, and 85% at 1% sucrose. While, the percentage reduction of root number, root fresh mass, root dry mass, respectively, were 9%, 50%, and 60% at 2% sucrose and 37%, 70%, and 80% at 1% sucrose (table 1). Correlation analysis for the relationship of different growth parameters showed that significant correlation existed among each other in response to different sucrose level.
The growth of excised cells, tissues, and organs in vitro depends on the supply of various metabolites and growth factors in the nutrient medium. Although many recent studies have dealt with the effects of growth regulators on the development of cultures, very little is known about the control exerted by carbohydrates. It is widely accepted that cultures of explants require an exogenous source of energy and carbon skeleton (15) and that sucrose has been the most effective, although some exceptions to this rule exist (27). Most nutrient media used for tissue cultures contain sucrose, usually at concentrations of 2 to 3.4% (3). The carbohydrate source contributes to the growth of shoots and roots due to its acts on cell expansion and proliferation and consequently increase the explant weight and volume (11, 28). However, high sugar concentrations may inhibit the plantlet growth due to osmotic stress in the medium (15, 17, 26). In vitro grown plants are devoid of photosynthesis so they need a readily available source of carbon which is usually provided in the medium at 2-3.4%. However optimum level of sucrose varies for different crops and even species (10). The reduction in chlorophyll content in in vitro plants may reduce photosynthetic ability by decreasing light absorption. The reduction in photosynthetic pigments due to carbohydrate addition in the medium has already noted (18).These authors suggested that lower sucrose concentrations may stimulate the chlorophyll production in in vitro potato plantlets. Plants cultivated on medium containing low or without sugar may have better photosynthetic rates in vitro when they are compared with plants grown on medium with sugar, carbon source additions to the medium in low concentrations are recommended (7).
In the second experiment, different concentrations of agar in MS medium were used for growth of single node explants. All tested traits were found to be effected by agar concentration. Decreasing agar level in the medium less than the normal used concentration (0.7%) in MS medium showed to some extent gradual increase in most parameters tested (table 2), which was clear in shoot and root mass. The percentage increase of shoot fresh mass, shoot dry mass, root fresh mass and root dry mass were 13%, 20%, 38%, and 70% at 0.5% agar respectively compared to the normal concentration used in MS medium (table 2). Low agar level has been reported to provide a poorly gelled medium that facilitates adequate contact between the plant tissue and the medium resulting in promoted shoot and root proliferation in several culture systems on account of faster uptake of minerals and plant regulators and better absorption of water (4,11, 16). There are many examples where better shoot growth and rooting have been observed in medium containing low agar level or liquid medium. The filter paper support provided in liquid medium gave better anchorage owing to its porosity that facilitated increased absorption throughout its surface area (21). The range of agar concentrations tested (from 0.5 to 0.7%) demonstrated that agar level of 0.5-0.6% to some extent were effective in promoting shoot and root development and growth. Nevertheless, low levels (0.5%) caused some cases of vitrification. Some researchers have reported that the leaves or leaf explants cultured at low agar levels were able to uptake more water from the medium and, therefore, exhibited much higher vitrification (14, 22). On the other hand, higher agar concentrations may result in a decrease in growth as agar would increase the solidness of the medium from which nutrient uptake would be limited. Based on our experimental results and observations, we can suggest that 0.5% agar is the most suitable concentration for single node explants culture of potato cv. Rio Grande.
Table 1: Effects of sucrose concentration on growth of potato plantlets
| Sucrose (%) | Shoot length (cm) | Leaflets number (No.) | Shoot fresh mass (mg) |
Shoot dry mass (g) |
Root length (cm) | Root number (No.) | Root fresh mass (mg) |
Root dry mass (mg) |
| 1 | 3.9 a | 4.05a | 140a | 6a | 7.75a | 6.2a | 30a | 10a |
| 2 | 5 a | 4.1a | 190a | 10a | 6.85a | 8.95b | 50a | 20a |
| 3 | 8.15 b | 5.55b | 300b | 40b | 5.55a | 9.8b | 100b | 50b |
Means with the same letters in a column are not significantly different from each other according to Duncan test at P˂5%.
Table 2: Effects of agar concentration on growth of potato plantlets
| Agar (%) | Shoot length (cm) | Leaflets number (No.) |
Shoot fresh mass (mg) |
Shoot dry mass (mg) |
Root fresh mass (mg) |
Root dry mass (mg) |
| 0.5 | 2.85a | 4.4a | 160a | 12a | 29a | 17a |
| 0.6 | 2.9a | 5.3a | 144b | 10a | 26b | 9b |
| 0.7 | 2.85a | 4.2a | 142b | 10a | 21b | 10b |
Means with the same letters in a column are not significantly different from each other according to Duncan test at P˂5%.
References
[1] Ahmed, K.U., and Rashid M. 1990. Eradication of potato virus X (PVX) by thermotherapy and meristem tip culture. J. Plant Pathology. 5 (1-2); 65-69.
[2] Badr A., Angers P., and Desjardins Y. 2011. Metabolic profiling of photoautotrophic and photomixotrophic potato plantlets (Solanum tuberosum) provides new insights into acclimatization. Plant Cell Tiss. Org.107: 13–24.
[3] Cabasson C., Ollitrault P., Cote, N., Michaux D., Dalnic R., and Teisson C. 1995. Characteristics of Citrus cell cultures during undifferentiated growth on sucrose and somatic embryogenesis on galactose, Physiol. Plant. 393-464.
[4] Casanova E., Moysset L., and Trillas M. 2008. Effect of agar concentration and vessel closure on the organogenesis and hyperhydricity of adventitious carnation shoots. Biol. Plant. 52: 1-8.
[5] FAO. 2014. Year book of aquaculture department. Food and agriculture organization of the united nations, Rome - Italy.
[6] Fuents S., Calheiros M., Manetti-Filho J., and Vieira L. 2000. The effect of silver nitrate and different carbohydrate sources on somatic embryogenesis in Coffea canephora. Plant Cell Tiss. Org. Cult. 50-60.
[7] Fuentes G., Talavera C., Opereza C., Desjardins Y., and Santamaria J. 2005. Exogenous sucrose can decrease in vitro photosynthesis but improve field survival and growth of coconut (Cocos nucifera L.) in vitro plantlets. In vitro Cell, Dev. Biol-Plant. 41(1):69-76.
[8] Gamborg, O., and Phillips G. 1995. Plant cell, tissue and organ culture. Spring- Verlag Berlin Heidelberg. 1- 21.
[9] George E. 1993. Plant propagation by tissue culture, Part 1: The Technology, Exegetics Ltd. Edington, England. 1-43.
[10] George E., Hall M., and Deklek K. 2008. Plant propagation by tissue culture. Dordrecht, Netherlands 3rd Edition Springer. 1- 50.
[11] Gurel S., and Gulsen Y. 1998. The effects of different sucrose, agar and pH levels on in vitro shoot production of almond ( Amygdalus communis L). Turk. J. Botany. 22:363-373.
[12] Hazarika B. 2003. Acclimatization of tissue cultured plants. Current Sci. 85:1705–1712.
[13] Hazarika B. 2006. Morpho-physiological disorders in vitro culture of plants. Sci. Hort. 108: 105–120.
[14] Hussey G. 1986. Vegetative propagation of plants by tissue culture. In: Yeoman M.M. (ed.), Plant Cell Culture Technology, Blackwell Scientific Publications, Oxford.
[15] Jo E., Tewari R., Hahn E., and Paek K. 2009. In vitro sucrose concentration
affects growth and acclimatization of Alocasia amazonica plantlets. Plant Cell Tiss.Org. 96: 307–315.
[16] Klimaszwaska K., Bernier M., Cyr D., and Sutton B. 2000. Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic culture of Pinus strobus L., In Vitro Cell Dev. Biol-Plant. 36: 279-289.
[17] Martins J., Moacir P., Adalvan D., and Suelen F. 2015. Effects of salts and sucrose concentrations on in vitro propagation of Billbergia zebrina (Herbert) Lindley (Bromeliaceae). Aust. J. Crop Sci. 9(1):85-91.
[18] Mohamed M., and Alsadon A. 2010. Influence of ventilation and sucrose on growth and leaf anatomy of micropropagated potato plantlets. Sci Hort. 123 (3): 295–300.
[19] Murashige T., and Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.
[20] Pierik R. 1978. In vitro culture of higher plants, Martinus Nijhoff, Dordrecht, 1-24.
[21] Puchooa D., Purseramen P., and Rajbally B. 1999. Effects of medium support and gelling agent in the tissue culture of tobacco (Nicotiana tabacum). Sci. Technol. 3:129-145.
[22] Rugini E., and Verma D. 1983. Micropropagation of a difficult-to propagate almond (Prunus amygdalus) cv. Batsch. Plant Sci. Let. 28, 273-281.
[23] Scholten H., and Pierik R. 1998. Agar as gelling agent differential biological effects in vitro, Sci. Hortic. 77: 109-116.
[24] Selby R., and Harvey B. 1989. The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.] New Phytologist. Vol. 113 (2) 203-210.
[25] Simko I. 1994. Sucrose application causes hormonal changes associated with potato tuber induction, J Plant growth Reg. 13-73.
[26] Siwach P., Grover K., and Gill A. 2011. The influence of plant growth regulators, explant nature and sucrose concentration on in vitro callus growth of Thevetia peruviana Schum. Asian J. Biotechnol. 3(3):280-292.
[27] Van Quy L., Samson G., and Desjardins Y. 2001. Opposite effects of exogenous sucrose on growth, photosynthesis and carbon metabolism of in vitro plantlets of tomato [L. esculentum Mill.] grown under two levels of irradiances and CO2 concentration. J. Plant Physiol. 158: 599–605.
[28] Wang L., and Ruan Y. 2013. Regulation of cell division and expansion by sugar and auxin signaling. Front Plant Sci. 4:1-9.