Growth Improvement of Potato Plants Produced from Tissue Culture

Middle East Journal of Agriculture Research ISSN 2077-4605 Volume : 05 | Issue : 04 | Oct.-Dec. | 2016 Pages: 666-671 Growth Improvement of Potato Plants Produced from Tissue Culture M. M. Khalil, A. M. H. Abd El Aal and M. M. Samy Potato and Vegetatively Propagated Vegetables Department, Horticulture Research Institute, A.R.C., Giza, Egypt Received: 25 November 2016 / Accepted: 30 December 2016 / Publication date: 31 December 2016 ABSTRACT The production of strong healthy plant material ex vitro is essential for an efficient seed potato production programs. Current study shows an applicable method for enhancing performance of potato plantlet subsequent in vitro phase. Using of ventilation and omitting sucrose from the nutrient medium (photoautotrophic culture) resulted in strong plantlets with higher fresh and dry weight performed better during acclimatization. Furthermore, Using of culture media contain macronutrients based on potato leaves macronutrients concentrations produced vigorous plantlets. Varietal differences were recorded between the two tested varieties Cara and Diamant. To obtain healthy start material for seed potato program this study recommend the use of in vitro photoautotrophic culture and modified nutrient media based on potato leaf macronutrient concentrations. Key words: Potato, Tissue culture, photoautotrophic, mineral nutrition Introduction Potato (Solanum tuberosum L.) is one of the most important vegetable crops in Egypt. In 2014 total cultivated area reached about 410 thousand Feddan (172 thousand hectare) produce around 4.6 million ton with average 11.26 ton per Feddan (FAOSTAT, 2016). Seed potatoes in Egypt fall into three categories: imported certified seeds, locally multiplied (in open field) certified seeds, and locally produced uncertified seeds. In 2013 Egypt imported about 184.5 thousand tons potatoes from Europe cost 144.7 million US$ (FAOSTAT, 2016). It is of great importance to establish a national program for seed potato production to save the foreign currency and to maximize the production in the three cultivation seasons using higher grade seeds produced by means of tissue culture techniques. In vitro treatments affect the subsequent growth of minitubers in greenhouses (Lommen, 1999; Tadesse et al. 2001 and Milinkovic et al. 2012). The photoautotrophic micropropagation (micropropagation with no exogenous organic components, i.e. sugar) has advantages over the conventional one with culture media containing sugar since it has less necessity for keeping aseptic environment, higher growth rates, less physiological disorders, higher survival of plantlets and lower production costs (Kozai and Smith, 1995). Furthermore, Liefert et al. (1995) stated that researchers often tended to try one nutrient medium when they attempted to in vitro culture plant species mostly MS media (Murashige and Skoog, 1962) and if the growth was satisfactory it considered the suitable medium for this species without comparing to other media compositions for this reason it is not affirmed if the published culture media ensure the maximum growth of these species. Moreover, Terrer and Tomas (2001) obtained higher proliferation rates, height, number of leaves and lower hyperhydericity in six peachalmond hybrids when cultured on media contain macronutrients corresponding to leaves concentrations in the period of maximum growth comparing with MS media. Also, Gonçalves et al. (2005) reported that in vitro rooting and shoot growth of carob tree was enhanced on culture medium based on leaves mineral analysis. Likewise, Bouman and Tiekstra (2005) improved in vitro propagation of Gerbera and Cymbidium on media adapted according the macronutrients to the elemental composition of adult leaves. In Egypt many studies have been conducted on propagation of potato by tissue culture techniques but did not lead to develop a complete program for potato seed production locally in spite it has become a principle technique in potato seed production in developed countries and many developing countries. There is a lake of literatures dealing with improving growth of plantlets Corresponding Author: M. M. Khalil, Potato and Vegetatively Propagated Vegetables Department, Horticulture Research Institute, A.R.C., Giza, Egypt E-mail: [email protected] 666 Middle East J. Agric. Res., 5(4): 666-671, 2016 ISSN 2077-4605 produced in vitro and enhancement of growth during acclimatization and subsequent growth in vivo. This study tries to manipulate conditions in vitro to produce a high quality starting material for seed potato production program. For this purpose this study investigates the effect of ventilation and modifying macronutrients concentration in culture media on production of plantlets of two potato varieties. Materials and Methods Stem cutting explants from meristem cultures of two potato (Solanum tuberosum L.) varieties Cara and Diamant were used. Meristem tip culture media was modified MS (Murashige and Skoog, 1962) salts and vitamins media (Cassion Laboratories Inc. USA ) supplemented with 10 mg/l Adenine sulfate, 5 mg/l calcium pantothenate, 1.0 mg/l GA3, 30 g/l sucrose and 7 g/l agar. The pH was adjusted to 5.7 before autoclaving at 1.45 Kg/cm2 for 20 min. Five cm length sprouts were separated then washed under running tap water for 30 min. Sprouts were surface sterilized with 10 % Chlorex (5.25% NaOCl) with tow drops of surfactant (tween 20) and agitation for 20 min. After that sprouts were rinsed three times with sterile distilled water. Meristem tip were desiccated under binocular in laminar airflow. The obtained shoots were subcultured every 4 weeks on the same Modified MS media without growth regulators. The obtained shoots were the source of stem cuttings. For conducting in vitro experiment stem cutting of the two varieties (Cara and Diamant) were cultured on four culture media and cover treatments: 1. 2. 3. 4. MS+ 30 g sucrose with normal polycarbonate cover (Mixotrophic). MS without sucrose with ventilated polycarbonate cover (Autotrophic, Fig 1). Potato media (PM, Table 1) with sucrose with normal polycarbonate cover (Mixotrophic). Potato media (PM) without sucrose with ventilated polycarbonate cover (Autotrophic). Fig. 1: The ventilated polycarbonate cover The potato media (PM) composed of macronutrients calculated based on factor between potato leaves maximum content (Kolbe and Stephan-Beckmann, 1997) and MS macronutrient. The factor generated from dividing total concentration of macronutrients in MS media (in mg) by the total leaf macronutrients concentration (in percentage) i.e. 1818.83/18.2=99.94. Multiplying this factor (99.94) by each macronutrient concentration in potato leaf we obtained new macronutrients concentration for potato media (Terrer and Tomas, 2001). Table 1 illustrates the concentration of macronutrients of potato media comparing with MS macronutrients. The salts used as source for macronutrients indicated in Table 2. The ventilated covers were obtained by adhering three disk filters (TC-FM 19 mm adhesive PTFE filter membrane, pore size 0.2 µm - Shanghai Zeshine Equipment Co., Ltd., China) on three holes on the usual polycarbonate covers used in plant tissue culture laboratories (Fig 1). Five explants were cultured in each jar. Each treatment was replicated five times with 10 jars per replicate. The experiment statistical design was factorial analysis in complete randomized design. Data of plantlet length, number of leaves and rooting were recorded after 4 weeks from culture. The in vitro produced plants were acclimatized by transferring the plantlets into small plastic cups filled with peat moss and vermiculate (1:1; V/V) where they covered with poly ethylene film that gradually removed after 3 weeks. Data of survival percentage were recorded. Data was statistically analyzed using Statistix 8 software. 667 Middle East J. Agric. Res., 5(4): 666-671, 2016 ISSN 2077-4605 Table 1: The applied macronutrient concentrations in MS media and potato media. Macronutrient Nitrogen Potato leaf (%) 7.1 MS media (mg/l) 839.96 Potato media (mg/l) 709.54 Potassium 5.2 783.66 519.66 Phosphorous 0.8 38.72 79.95 Calcium 4.3 120.01 429.72 Magnesium 0.8 36.47 79.95 Total 18.2 1818.82 1818.82 Table 2: Salts and concentrations used as source for macronutrients. Salt KNO3 NH4NO3 KH2PO4 CaCl2.2H2O Ca(NO3)2.4H2O Mg SO4.7H2O Molecular Weight 101.1 80.09 136.09 147.02 236.1 246.5 MS media (mg/l) 1900 1650 170 440 ----370 Potato media (mg/l) 1083 745 351 ----2530 811 Results and Discussion Table 3 shows the effect of variety, media and ventilation on potato plantlets shoot length, leaves number, fresh and dry weight after 4 weeks of in vitro culture. Table 3: Effect of variety, media and ventilation on potato plantlet growth in vitro after 4 weeks of culture. Variety Media* Nutrition Cara MS Mixotrophic Autotrophic Mixotrophic Autotrophic Mixotrophic Autotrophic Mixotrophic Autotrophic PM Diamant MS PM Cara Diamant LSD 0.05 LSD at 0.05** MS PM LSD at 0.05 Shoot length (cm) leaves No° 6.9 3.7 7.1 4.2 7.1 2.0 3.1 1.0 0.9 5.5 3.3 0.5 4.9 3.8 0.5 6.4 5.8 5.0 5.6 7.0 3.8 3.6 4.0 1.2 5.7 4.6 0.6 5.7 4.6 0.6 Mixotrophic Autotrophic LSD 0.05 Shoot Fresh Weight (mg) 117.4 165.8 83.4 153.8 109.3 117.4 122.2 126.0 NS 130.1 118.7 NS 127.5 121.4 NS Shoot Dry Weight (mg) Dry Matter (%) 12.6 13.4 9.0 12.4 13.3 14.0 13.8 17.0 NS 11.9 14.5 1.5 13.3 13.1 NS 10.7 7.9 10.8 8.6 12.2 12.5 11.2 13.5 NS 9.6 12.4 1.9 10.8 11.1 NS 6.0 5.5 108.1 12.2 2.7 4.8 140.8 14.2 0.5 0.6 28.3 1.5 Cara MS 5.3 6.1 141.6 13.0 PM 5.6 5.3 118.6 10.7 Diamant MS 4.6 5.4 113.3 13.6 PM 2.1 3.8 124.1 15.4 LSD at 0.05 0.7 NS NS 2.2 Cara Mixotrophic 7.0 5.7 100.4 10.8 Autotrophic 4.0 5.7 159.8 12.9 Diamant Mixotrophic 5.1 5.3 115.7 13.5 Autotrophic 1.5 3.9 121.7 15.5 LSD at 0.05 NS 0.9 40.0 NS MS Mixotrophic 7.0 6.7 113.3 12.9 Autotrophic 2.9 4.8 141.6 13.7 PM Mixotrophic 5.1 4.3 102.8 11.4 Autotrophic 2.6 4.8 139.9 14.7 LSD at 0.05 0.7 0.9 NS NS *MS: Murashige and Skoog nutrient media; PM: Potato nutrient media modified media based on potato leaves concentration of macronutrients.**LSD at 0.05: least significant difference at 0.05. 668 11.3 10.6 NS 9.3 9.9 12.3 12.4 NS 11.0 8.2 11.7 13.0 2.7 11.4 10.2 11.2 11.1 NS Middle East J. Agric. Res., 5(4): 666-671, 2016 ISSN 2077-4605 Cara recorded higher values of shoot length and leaves number than Diamant (Table 3Fig.2). MS gave higher shoot length and leaves number than PM. The presence of sucrose in media enhanced shoot length and leaves number. Although autotrophic treatments (ventilated covers without sucrose in media) produced shorter shoots with smaller distance between nodes. Autotrophic cultures attained higher shoot fresh and dry weight. Moreover, omitting of sucrose from media resulted in poor rooting (Fig.2). According to shoot dry weight, Diamant gave higher values than Cara this could be attributed to the greater dry matter percentage in Diamant. Furthermore, the interaction between cultivars and nutrition was significant hence Cara plantlets grown in ventilated vessels on medium free of sucrose gained the highest shoot fresh weight. These findings are in accordance with those obtained by Pruski et al (2002) who reported that potato plantlets grown photautotrophically were shorter with more nodes and higher in dry weight than mixotrophic cultures. Also, Kozai et al. (1988) and Kozai et al. (1992) reported that photoautotrophic micropropagated potato plantlets had higher fresh and dry weight. On the other side, Mohamed and Alsadon (2010) mentioned that potato plantlets grown in ventilated vessels were significantly shorter, lower fresh weight and higher dry weight than those in non-ventilated vessels. However, Pruski et al (2002) suggested that photoautotrophic culture of potato could be considered when high sterility levels not available. Furthermore, photoautotrophic enhance quality, size of produced plantlets. Concerning the chemical composition of nutrient medium, no significant differences between MS and PM media were observed although nitrogen concentration was reduced in PM which could be of economic importance in media costs. Fig. 2: effect of macronutrients on growth of the Cara (left) and Diamant (right) varieties under photoautotrophic or photomixotrophic culture: 1. MS+ 30 g sucrose with normal polycarbonate cover. 2. MS without sucrose with ventilated polycarbonate cover. 3. Potato media (PM) with sucrose with normal polycarbonate cover. 4. Potato media (PM) without sucrose with ventilated polycarbonate cover. 669 Middle East J. Agric. Res., 5(4): 666-671, 2016 ISSN 2077-4605 After 4 weeks of acclimatization performance of plantlets grown photautotrophically were healthier in terms of number and size of leaves (Fig 3). The obtained results agreed with those obtained by Pérez-Jiménez et al. (2015) who stated that growth of plantlets under photoautotrophic conditions reduces loss of plantlets, increase water use efficiency and photosynthesis resulting in increasing plantlet survival after acclimatization. This could be attributed to the difference between growth conditions hence in photomixotrophic culture high relative humidity, low exchange of gases, low photosynthetic photon flux and the presence of sugar in the medium suppress photosynthesis causing morphological and physiological abnormalities i.e., leaf malfunction of stomata, thin unorganized palisade and mesophyll layer, less wax deposition (Afreen, 2005). Fig. 3: Growth of Diamant (left) and Cara (right) plantlets after 2 weeks of acclimatization Cara MS without Sucrose Cara PM without Sucrose Fig. 4: Growth of Cara plantlets grown photautotrophically after 4 weeks of acclimatization 670 Middle East J. Agric. Res., 5(4): 666-671, 2016 ISSN 2077-4605 As a conclusion it’s recommended to grow potato plantlets during in vitro phase under conditions close as possible to conditions ex vitro. The results of the current study suggest the use of photoautotrophic culture as an improvement tool of gases environment in vitro to produce a strong and healthy plantlets. Also, the obtained results recommend adjusting the nutrient media according to potato leaf content. The obtained plantlets represent a good starting material for seed potato production program. References Afreen, F., 2005. Physiological and Anatomical Characteristics of in Vitro Photoautotrophic Plants. In: Kozai, T., Afreen, F., Zobayed, S.M.A. (Eds.), Photoautotrophic (Sugar-Free Medium) Micropropagation as a New Micropropagation and Transplant Production System. Springer Netherlands, Dordrecht, pp. 61–90. Bouman, H. and A. Tiekstra, 2005. 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