Optimization of in vitro regeneration of multiple shoots from hypocotyl sections of cotton (Gossypium hirsutum L.)

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1 African Journal of Biotechnology Vol. 3 (3), pp , March 2004 Available online at ISSN Academic Journals Full Length Research paper Optimization of in vitro regeneration of multiple shoots from hypocotyl sections of cotton (Gossypium hirsutum L.) J. P. Ouma 1 *, M. M. Young 2, and N. A. Reichert 2 1 Agronomy Department, Egerton University, P.O. Box 536, Njoro, Kenya. 2 Department of Plant and Soil Sciences, 117 Dorman Hall, P.O. BOX 9555 Mississippi State University MS 39762, USA. Accepted 1 January 2004 A direct shoot regeneration protocol was optimized for cotton (Gossypium hirsutum). Two cultivars of cotton, Delta pine 50 (DP50) and Stoneville 474 (STV474) were used to study the effects of thidiazuron (TDZ), naphthalene acetic acid and silver nitrate (AgNO 3 ) on shoot regeneration from hypocotyl explants excised from 14 day-old seedlings cultured in vitro. The best treatment for formation of adventitious shoots in DP50 was the treatment containing mg l -1 TDZ, 0.01 mg l -1 NAA and 5.1 mg l -1 AgNO 3, while treatment containing 0.08 mg l -1 8 TDZ, 0.01 NA mg l -1 A and mg l AgNO 3 was optimum for the formation of adventitious shoots for STV474. Key words: Thidiazuron, silver nitrate, direct organogenesis, media optimization, cotton, Malvaceae. INTRODUCTION The use of biotechnological tools such as biolistics and Agrobacterium-mediated transformation, require as a prerequisite plant regeneration protocols that are genotype-independent, efficient, and which do not yield somaclonal variant (Firoozabady et al., 1987; Gould and Cedeno, 1998; McCabe and Mattinell, 1993). Cotton is known to be recalcitrant in in vitro regeneration systems. A number of regeneration protocols have been developed for cotton using various explants and through manipulation of media composition. Somatic embryogenesis in cotton has been reported (Trolinder and Grodin, 1987; Firoozabady and DeBoer, 1993). However, response was genotype specific and may also produce somaclonal variants where a callus phase is involved. Explants with pre-existing meristems have also been used to induce multiple shoots in vitro and regenerate whole plants (Agrawal et al., 1997; Gupta et al., 1997; Hemphill et al., 1998; Morre et al., 1998; Hazra et al., 2000). These included cotyledonary nodes with or without cotyledons and shoot tip, shoot tips alone, apical meristems and whole or split cotyledonary node explants. *Corresponding author. Tel (254) Fax (254) jpam4_2001@yahoo.com. Early reports of use of hypocotyls explants to induce multiple shoots in cotton were reported by Umbeck et al. (1987) and Firoozabady et al. (1987). Firoozabady et al. (unpublished), attributed the formation of non transformed calli to the use of hypocotyls explants, concluding that hypocoty tissue was not ideal for developing a high-efficiency cotton regeneration system for Agrobacterium-mediated transformation. Pandey and Bansal (1989), found a reduced callus formation from hypocotyls explants in cowpea (Vigna sinensis L.). Other studies that have utilized hypocotyls include somatic embryogenesis in cowpeas (Amitha and Reddy, 1996; Pellengrineschi, 1997).). Hemphill et al. (1998), observed an occasional shoot development from cotton hypocotyls segments, suggesting that they have potential for direct shoot organogenesis. The composition of the culture medium and the gaseous environment are important factors in regeneration of shoots in vitro. Varying amounts of ethylene is released in culture vessels during in vitro regeneration (De Proft et al., 1985). In recalcitrant plant species ethylene accumulation within the culture vessel has been associated with low in vitro regeneration efficiency (Chi and Pua, 1989; Chi et al., 1990; Mohuiddin et al., 1997). Addition of silver nitrate to the culture medium increased shoot regeneration response from

2 170 Afr. J. Biotechnol. cucumbers (Chi and Pua, 1989; Mohuiddin et al., 1997). Cytokinins stimulate shoot proliferation in tissue culture. Benzyladenine (BA) is an important cytokinin used in promotion of shoot regeneration. A number of studies have reported that thidiazuron (TDZ) which is a thiadiazolyurea derivative is more potent than BA in in vitro regeneration of shoots in dicotyledonous species (Singh et al., 2003). A study on Kenaf (Hibiscus spp.) and cotton (Gossypium hirsutum) showed that addition of TDZ and silver nitrate to shoot initiation medium increased the number of multiple shoot buds formed on the proximal end of hypocotyls explants (Young MM and Reichert NA, unpublished results). The objective of this study was therefore to optimize the protocol for multiple shoot regeneration based on a modified Murashige and Skoog basal medium and using thidiazuron and Naphalene acetic acid as cytokinin and auxin source, and silver nitrate as an ethylene-action inhibitor. MATERIALS AND METHODS Seed sterilization procedure De-linted cotton seeds of cultivar DeltaPine50 (DP50) and Stoneville 474 (STV474) were surface sterilized by immersing in 70% ethanol and shaking for 5 min in a rotary shaker at 200 rpm. The alcohol was decanted under sterile conditions and seeds rinsed with sterile, deionized water three times with 1 min shaking between rinses. The seeds were further treated with 0.5% sodium dodecylsulphate (SDS) and 25% commercial bleach (5.25% sodium hypochlorite) for 25 min in a rotary shaker at 200 rpm. Seeds were rinsed again with deionized sterile distilled water three times with 1 min shaking between rinses. Media preparation The media consist of 3% glucose, full strength Murashige and Skoog salts, modified MS vitamins (DM vitamins consisting of 0.5 mg/l Pyridoxine-HCL, 10.0 mg/l Thiamine-HCL, 0.5 mg/l Nicotinic acid, 2mg/l Glycine, and 100mg/l Myo-inositol), and solidified with 0.8% phytagar. The ph was stabilized at 5.8 before addition of phytagar. The medium was autoclaved for 35 min at a pressure of 1.1kg/cm 2 a temperature of 124 C. After cooling, Cefotaxime (antibiotic) was added at 500 mg/l into the medium. The medium was poured into sterile petri-dishes 100 x 10 mm depth and allowed to solidify. Seed germination and experimental protocol Fifteen seeds were placed in each petri-dish, and sealed with Nescofilm (Karlan Research Products Corp., Santa Rosa, California). The petri-dishes were incubated in a growth chamber at 25±2 C, under 18/6 h. photoperiod for 7 days. Germinating seeds were transferred into germination medium contained in magenta boxes as above but not solidified with phytagar. The magenta boxes were lined with 4 layers of cheese cloth to provide support for the seedlings seedlings were placed in each magenta box which was then sealed with Nescofilm, and incubated at 25± 2 C at 18/6 h photo-period for 7 days. The 14 day old seedlings were excised without roots. Hypocotyl sections (1 cm length) were obtained from the seedlings by cutting at the base of the cotyledons. 10 hypocotyls explants were placed into shoot initiation medium contained in 100 x 15 mm petri-dish. Shoot initiation medium consisted of basal medium (3% glucose, MS salts DM vitamins, 0.8% phytagar, and ph 5.8). After autoclaving the medium, filter sterilized NAA, AgNO 3 together with TDZ were added to the cooled medium according to the treatments. The first experiment consisted of 6 TDZ rates (0.088, 0.175, 0.35, 0.70, 1.4, and 2.1 mg/l), 3 NAA rates (0.01, 0.05, and 0.1 mg/l) and AgNO 3 added at 10.2 mg/l. The second experiment consisted of varying levels of TDZ (0.088, 0.175, 0.35 mg/l), NAA (0.01, 0.05 mg/l), and AgNO 3 (5.1, 10.2, 20.4mg/l). Control medium consisted of 0.35mg/lTDZ, 0.1mg/lNAA, and 10.2 mg/l AgNO 3.The experiments were laid out as a completely random design with 20 explants per treatment. The experiments were repeated two times. Petri dishes containing 10 explants were sealed with Nescofilm and incubated for 6 weeks at 25± 2 C under a 16/8 h photoperiod. Radiation source was supplied by soft white fluorescence tubes. At 6 weeks, the cultures data were collected on number of responding explants. Response was defined in terms of presence of shoots. The site of origin of the shoots were also noted and classified as either from the distal or proximal end of the explant, from the side, middle or both middle and sides of the responding end. Explants forming shoots were trimmed at the base and placed in shoot elongation medium, composed of the basal medium supplemented with 0.1 mg/l BA and left to elongate for 6 weeks. Elongated shoots were lightly trimmed at the base and transferred into rooting media of various composition and incubated for 6 weeks. Rooted shoot were transferred into well-moistened potting mix [BM1 mix (Canadian sphagnum peat moss 75-85%, perlite 15-20%, Vermiculite 5-10%)] in 7.5 cm diameter pots. The pots were covered with clear polyethylene bags to provide 100% relative humidity. They were placed in an acclimatization room at 25±2 C, under 16/8 h photoperiod. Ventilation holes were made in the polythene cover after 7 days. The hole was subsequently made larger until the whole cover was removed at 14 days. Acclimatized plants were transferred to larger pots with timed-release fertilizer and allowed to grow to maturity. Statistical analysis was carried out using SAS (Cody and Smith1991). Means of transformed data were then squared and added to MSE of non-transformed data to give an overall mean (Steel and Torrie, 1980). RESULTS AND DISCUSSION The two genotypes used, STV474 and DP50 gave a similar response in shoot regeneration media containing variable concentrations of TDZ and NAA. The hypocotyls of both cultivars displayed a distinct polarity with adventitious shoots arising from the proximal end of the explant (Figure 1). From previous experiments (Young MM, Reichert NA, unpublished data), the response of cotton hypocotyl in tissue culture displays a distinct polarity. In cowpeas (Brar et al., 1999) and cucumbers (Mohiuddin et al., 1997) numerous shoot buds arose from the proximal end of hypocotyl explant showing that this phenomenon is common in dicotyledonous species. Adventitious shoot regeneration occurred at the proximal end of hypocotyls placed horizontally in culture (Figure 1a). Prior to the formation of shoot buds, the proximal end swelled, and then formed primordial buds with or without a callus phase depending on the medium (Figure 1b). However, only a few primordial buds developed into

3 Ouma et al. 171 Figure 1. Regeneration of adventitious shoots from hypocotyl explants of cotton. (a) Shoots developing from the proximal end of hypocotyls of cultivar STV474 on medium containing mg l -1 TDZ, 0.01 mg l -1 NAA and 5.1 mg l -1 AgNO 3 after 6 weeks in culture. (b) Hypocotyl explants of cultivar STV474 on control medium (0.35 mg l -1 TDZ, 0.1 mg l -1 NAA, and 10.2 mg l -1 AgNO 3 ) after 6 weeks in culture. (c) Hypocotyl explants of cultivar DP50 on medium containing mg l -1 TDZ, 0.01 mg l -1 NAA and 5.1 mg l -1 AgNO 3 showing adventitious shoot buds and leafy structures arising from the central region and sides of the proximal end of hypocotyls after 6 weeks in culture. a b c shoot buds, with some shoot buds developing into shoots 1 to 2 mm in length within 6 weeks in culture (Figure 1c). The rate of shoot bud development differed between locations on the proximal end of responding hypocotyls. Those shoot buds developing slightly to the side of the swollen hypocotyl were generally more developed and elongated than those in the central region of the same hypocotyl, particularly in cultivar STV474. It is possible that these shoot buds arose from preexisting axillary bud meristems at the junction of the cotyledon and hypocotyl, thus explaining their rapid morphological development. Each hypocotyl explant formed 1 to 2 shoots at the center with or without other shoots arising from the side of the explant. A number of the shoot buds at the center of the proximal end of the hypocotyl did not continue to form shoots. It would appear that the well-developed shoots suppressed further development of shoot buds. When these shoots were excised from the explant and the remaining hypocotyl containing shoot primordial and shoot buds subcultured in shoot initiation or elongation medium, those hypocotyls in shoot initiation began to form cream-colored friable callus which eventually covered the whole explant, suppressing further development. The highest proportion of explants forming adventitious shoots was obtained with media containing TDZ from to mg l -1 (Table 1). At higher concentrations, the explants responded by producing some buds or shoot clumps as well as excessive amounts of brown exudates. This suppressed further development of buds. Buds formed on media containing high TDZ concentration also exhibited signs of hyperhydricity. At to mg l -1, the rate of shoot initiation was higher, the numbers of shoots elongating per explant in the shoot initiation media were greater, giving 1 to 2 shoots/explant at the lower TDZ levels compared to media with higher TDZ concentrations. In the control medium, many buds appeared as swellings, but only 1 to 2 elongated when transferred to a shoot elongation medium containing 0.1 mg l -1 BA for 6 weeks. The optimum range for both cultivars was to mg l - 1 TDZ in combination with 0.01 to 0.1 mg l -1 NAA and 10.2 mg l -1 AgNO3. After the optimization of TDZ and NAA rates, an experiment was conducted to determine the effect of AgNO 3 concentration on the initiation and regeneration of shoots from hypocotyls explants. Multiple shoot formation in both DP50 and STV474 were not significantly different at p<0.05 among media treatments (Table 2). The trend however was that relatively more shoots/treatment were obtained in the treatment containing mg l -1 and 0.01 mg l -1 TDZ and NAA, respectively, in combination with 5.1 mg l -1 AgNO 3. Lower levels of TDZ induced relatively fewer shoot buds, but these developed rapidly into shoots (Figure 1a). In contrast, media containing 0.35 mg l -1 TDZ had more primordial visible but only a few developed into shoots. Nielsen et al. (1993), showed in Miscanthus sinensis that

4 172 Afr. J. Biotechnol. Table 1. Adventitious shooting response of hypocotyl explants of STV474 and DP50 on thidiazuron and Naphthalene acetic acid media at 30.2 mgl -1 AgNO 3. Treatment no. Treatment combination (mg/l) % Multiple shoots TDZ NAA STV474 DP a a a ab bcd abcd abcd abcd cd cd bc d ab abcd cd cd abc bcd cd bcd d bcd d d abcd abcd abc bcd cd bcd d d cd cd cd d Means in the same column followed by the same letter are not significantly different at p< Table 2. ANOVA table for the effect of media composition on multiple shoot regeneration in cotton genotypes STV474 and DP50 at 6 weeks in culture. Source DF Genotype DP50 Genotype STV474 MS Pr>F MS Pr>F Media Treatments ns ns Blocks ns s shoot size was highly negatively correlated with the number of shoots on a shoot cluster and that at higher TDZ concentrations the shoot size was reduced. In chile pepper, 2 mg/l of TDZ induced multiple shoots but these were small and did not elongate much (Hyde and Phillips, 1996). The incorporation of 0.01, 0.05 and 0.1 mg l -1 NAA in shoot regeneration medium did not have a significant effect on the number of explants forming adventitious shoots in DP50. There was slight inhibition of shoot regeneration at 0.1 mg l -1 NAA. In STV474, 0.01 mg l -1 NAA was superior in adventitious shoot regeneration than those containing 0.05 and 0.1 mg l -1 NAA. This could be due to a higher level of endogenous auxins in the tissues of STV474. For cultivars STV474 and DP50, there was no difference in the three levels of AgNO 3 for the formation of adventitious shoots from explants. The presence of AgNO 3 in regeneration media has been shown to improve shoot regeneration from cotyledon and hypocotyl explants of a number of dicotyledonous species, some which show recalcitrance in tissue culture (Chi et al., 1990; Hyde and Phillips, 1996; Mohiuddin et al., 1997). For the two cultivars used in this experiment, the rates of silver nitrate used seem optimal. These results demonstrate that for both STV474 and DP50, the optimum medium for regenerating adventitious shoots through direct organogenesis consist of basal medium which is supplemented with to mg l -1 TDZ, 5.1 to 20.4 mg l -1 AgNO3 and 0.01 to 0.05 mg l -1 NAA. The best treatment for formation of adventitious shoots in DP50 was treatment 2, containing mg l -1 TDZ, 0.01 mg l -1 NAA and 5.1 mg l -1 AgNO 3. The optimum medium requirement for the formation of adventitious for cultivar STV474 was treatment 8, containing mg l -1 TDZ, 0.01 mg l -1 NAA and 10.2 mg l -1 AgNO 3. ACKNOWLEDGEMENTS The Authors gratefully acknowledge receipt of grant funds from Monsanto Company. We also thank Dr. Wallace who provided seed cotton and Dr. Patrick Gerard

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