Studies on Propagation of Strawberry (Fragaria X ananassa Duch) by Tissue Culture

Abstract

This study was carried out at the tissue culture laboratory of the Department of Horticulture, College of Agricultural Studies, Sudan University of Science and Technology, Shambat, Khartoum North. The source of the plant material was an in vitro stock of strawberry (Fragaria X ananassa Duch.) cv. “Festival” plantlets. 1.5-2.0 cm long shoot terminals were used as explants for culture initiation and subsequent experimentations. Multiple shoots induction and plantlets regeneration were obtained from in vitro cultured shoot tips by sequential subculture after each harvest for experimentation. A modified MS medium containing the normal MS salt-strength plus 3% sucrose, 100 mg/l inositol, 10 ml/l vitamin stock, 0.1 mg/l BA, 0.03 mg/l NAA and 7 g/l agar was used as basal medium. Experiments were conducted to investigate the effect of some chemical components of culture medium and physical and environmental requirements on growth and development of in vitro cultured strawberry shoot tips. Growth responses (in terms of number and length of shoots, leaf number, as well as root number and length) to the various treatments tested were recorded after 6 weeks from culture initiation. The results showed that the normal MS-salt strength (1X) had the greatest effect on the various growth parameters measured. Shoot number and elongation, number of leaves and number of roots and root elongation were markedly promoted at 1X MS-salt strength. The two lowest MS salt strengths tested promoted root initiation. On the other hand, the 3/4X- MS nitrate strength {MS (3/4 NO3)} treatment had the greatest effect on the various growth parameters measured compared to the other concentrations tested. Increasing the concentration of phosphorous of the original MS medium by adding 127.5 mg/l KH2PO4 markedly increased shoot proliferation as well as number of leaves, number of roots and root elongation. Sucrose at 3% was optimal for in vitro culture of strawberry shoot tips giving higher values for all parameters measured compared to other concentrations tested. All inositol concentrations tested increased significantly shoot number over the control with progressive increases in shoot number as the concentration of inositol in the medium was increased up to a maximum of 100 mg/l and then declined sharply at the higher concentration tested. Shoot elongation, on the other hand, was non-significantly reduced over the control and was significantly inhibited by inositol concentrations higher than the lowest concentration of inositol tested. The results of an experiment conducted to determine the effects of different concentrations of BA on in vitro culture of strawberry shoot tip, with a 0.03 mg NAA/l held constant, showed that the lowest concentrations of BA tested increased the values of shoot number, and length, number of leaves and number of roots significantly over the control with significant differences between treatments. With increasing BA concentration above 0.5 mg/l shoot formation declined but was still higher than in the control. The effects of NAA concentrations on growth and development of in vitro cultured strawberry shoot tips, with BA held constant at 0.1 mg/l, revealed that the two lowest concentrations of NAA, (0.01 mg/l and 0.03 mg/l), tested promoted shoot proliferation, with no significant difference in shoot number between treatments and control. The highest concentration of NAA, (0.3 mg/l), tested significantly decreased shoot count denoting the failure of NAA at this concentration to induce shoot bud differentiation. This would probably be due to the augmentation of natural apical dominance by NAA, hence suppressing shoot formation. Both shoot and root lengths were suppressed by all NAA concentrations tested with significant differences over the control. Leave number was, however, unresponsive to all concentrations of NAA tested whereas root formation was promoted with the lowest concentration of NAA tested with significant difference over the control. In relation to the influence of extirpative chemicals which have been shown to exhibit growth regulators-like activity, (pesticide, herbicide and fungicide), the effects of Furidan concentrations revealed that the concentration of Furidan, (1.0 mg/l), tested significantly increased shoot number and length, number of root and length over the control. Leaves formation was suppressed by all Furidan concentrations tested. All Stroby concentrations tested significantly repressed shoot elongation, leave formation, root initiation and elongation over the control. Shoot proliferation was however, non-significantly increased by the lowest Stroby concentration tested relative to the control. On the other hand, leave number, root number as well as root length were largely unresponsive to the Seven levels tested. Shoot proliferation however, responded differently. The concentration of Seven (1.0 mg/l), tested significantly increased shoot number and reduced shoot elongation relative to the control. Neither root number nor root elongation were responsive for the Glyphosate levels examined; but these levels stimulated shoot proliferation and leaves formation and inhibited shoot elongation with significant difference relative to the control. The concentration of Glyphosate, (0.8 mg/l), tested significantly increased shoot number, elongation and number of leaves compared to the other concentrations tested. Higher concentrations of Glyphosate concentration suppressed shoot proliferation and number of leaves. All gum arabic concentrations tested inhibited shoot elongation, leaf number, root number and length relative to the control treatment. Significant differences between treatments on all measured growth responses were noted. Shoot number significantly increased relative to the control on medium containing 1.0 g/l gum arabic with significant difference among treatments. Concentrations higher than 1.0 g/l significantly reduced shoot proliferation, which was still higher than in the control. The evaluation of the effect of silver nitrate revealed that all silver nitrate concentrations tested in this study had no effect on shoot elongation but promoted shoot proliferation, leaves number, root number, and root length with significant differences among treatments. The highest number of shoots, leaves, roots and root lengths were obtained on medium containing 4.0 mg/l AgNO3 with significant differences among treatments, whereas the lowest values were obtained on medium containing high concentration (8.0 mg/l) AgN03. All CH concentrations tested responded positively on shoot proliferation and elongation and leaf number. The values of these growth responses increased with increases in CH concentration, reaching a maximum at 50 mg/l and then decreased. Neither root number nor root elongation affected by all CH concentrations tested. The highest number of shoot the longest shoots and the greatest number of leaves were obtained on medium containing 50 mg/l CH. With increasing CH concentration to 100 mg/l, the highest concentration tested, the values of shoot number shoot length, and number of leaves declined but were still higher than in the control. The lowest values of shoot number, elongation and leaves number were recorded on medium containing no CH. All concentrations of activated charcoal, (AC), tested significantly reduced shoot proliferation and increased shoot elongation, leaves number, root number, and root elongation over the control, with significant difference between treatments. The longest shoots, the greatest number of leaves and of roots and longest roots were recorded with AC at 1.0 g/l. The largest number of shoots was obtained on medium containing 0.0 g/l AC. The highest concentration of AC (2.0 g/l) significantly inhibited all parameters measured. The value of all parameters measured increased with decreasing pH value up to a maximum pH of 5.5 and then declined sharply at the pH 6.0. The highest values of shoot proliferation and elongation, leaves number, root number and root length were obtained at pH 5.5. The least values for all parameters measured were associated with the lowest and highest pH values tested. Root number and elongation seems to be highly sensitive to pH of the culture medium. The experiment conducted to study effects of physical medium supports on growth and development of strawberry shoot tip explants showed that “Ashmaik”, the date fibre, outperformed all other gelling and matrices supports tested giving significantly high values for most parameters measured. Shoot proliferation and elongation increased with decreasing light intensity. Leave number was significantly inhibited by dark incubation conditions; leave formation was obtained with cultures incubated under relatively high light intensity. Incubation under dark condition completely inhibited root initiation. The acclimatization and ex- vitro establishment of rooted plantlets were taken to the green house. Peat moss planting medium had the greatest effect on the various growth parameters measured compared to other planting media tested. The highest number of shoots, the longest shoots, the largest number of leaves and the longest roots were obtained at the peat moss potting medium.