Plant Regeneration from Protoplasts of Lilium Ledebourii (Baker) Boiss
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J. Agr. Sci. Tech. (2014) Vol. 16: 1133-1144 Plant Regeneration from Protoplasts of Lilium ledebourii (Baker) Boiss S. K. Tahami 1, E. Chamani 1*, and N. Zare 1 ABSTRACT For half a century, the limitations of obtaining cross-combinations in lilies because of the incompatibility and incongruity between different varieties have been known. Somatic hybridization is one of the most powerful tools for achieving distant interspecific hybrids. For this purpose, protoplast preparation is a first and important step in efficient system for the regeneration of plants from protoplasts. Protoplast isolation method was previously developed in Lilium ledebourii (Baker) Boiss. In this study, several valuable experiments were done based on completely randomized design with 3 replications and also each experiment was repeated twice. The results revealed that cell wall and colony formation were better in a liquid medium than those on a semi-solid medium. The highest plating efficiency (1.34×10 6 per gr FW) and callus formation was obtained by using a medium containing 1 mg L -1 2,4-D, 0.2 mg L -1 Kin and 2 g L -1 Yeast extract. Micro calli were formed after one month of culture. Many plantlets were formed on the calli after transfer of the proliferated calli to regeneration medium. The highest plantlet -1 regeneration (91.66%) was obtained by using a medium containing 0.5 mg L NAA, 1.5 mg L -1 BA. Means comparison revealed that the semi- solid MS medium containing 0.5 mg L -1 NAA and 1.5 mg L -1 BA had the highest percentage of regeneration (91.66%), bulb number (8.83), and length (0.7366 cm), root length (0.421cm) and leaf number (13.66) and length (0.5052cm). Keywords: Callus formation, In-vitro culture, Medium, Proliferated calli . INTRODUCTION irregular grazing and poaching (Azadi and Khosh-Khui, 2007). Somatic hybridization is one of the most Among various types of lilies, asiatic, oriental and L. longiflorum hybrids have powerful tools for achieving distant Downloaded from jast.modares.ac.ir at 14:04 IRST on Tuesday October 5th 2021 premium potentialities in the florist trade interspecific hybrids (reviewed in Evans, (Kumar et al ., 2006). Lilium ledebourii Boiss 1983). In lilies, efficient methods for isolating is one of the important ornamental and odorant large quantities of protoplasts from calli et plants. This plant has high adaptability to Iran initiated from bulb scale tissues (Simmonds environmental condition. It has a wide al ., 1979), pollen (Tanaka et al ., 1987), and applicability in floral industry as cut flower generative cells (Tanaka, 1988) have been and potted plant. Lilium ledebourii is grown in reported. Plant regeneration from protoplasts the eastern slopes of Alburz Mountains, where has been achieved (Mii et al ., 1994), and a it is now protected on a small area of degraded modified protocol for the regeneration of beech forest around Damash village, Khangah plants subsequently established (Godo et al ., et al of Ardabil province and Kalardasht 1996, 1998). Mii . (1994) first succeeded (Rechinger, 1989). Wild populations are in regenerating fertile plants derived from the currently at risk of rapid eradication because of protoplasts of Lilium formolongi through _____________________________________________________________________________ 1 Department of Horticultural Science, Faculty of Agriculture, University of Mohaghegh Ardabili, Ardabil, Islamic Republic of Iran. Corresponding author; [email protected] 1133 _______________________________________________________________________ Tahami et al. successful cell division in a solidified medium MATERIALS AND METHODS containing picloram. However, we were not able to obtain divided cells from our Protoplast from young leaves of L. preliminary culturing of protoplasts isolated ledebourii were isolated from leaf explants, from various kinds of oriental hybrid cultivars digested with different enzyme solutions and according to their protocol. Protoplasts can be also purified as described previously isolated from plant tissues or cultured cells. (Chamani et al ., 2012). The success of protoplasts isolation depends especially on the condition of the tissue and the combination of enzymes being used. The Protoplast Culture individual cell or tissue source may require special conditions for successful isolation or Protoplasts were cultured at a density of for culturing. Protoplasts isolated from cell 1×10 6 protoplasts ml -1. Two culture systems suspension cultures, generally, are readily were tested: liquid culture and semi-solidified obtained and usually regenerate into dividing culture. For the semi-solidified culture, cells at a reasonable frequency. Moreover, if protoplasts were plated as a thin layer on a MS the plant regeneration is possible from the cells medium containing 9% mannitol and of the suspension culture, the same is often solidified with 8 g L -1 agar in small petri true for the cells regenerated from the dishes. For liquid culture (MS without agar, protoplast derived from the culture (Tan et al ., with 9% mannitol), protoplasts were 1987). Leaf mesophyl cells of a wide range of suspended in 4 ml of media in small Petri plants also have been used as a protoplasts dishes (5.5 cm diameter). source with success by Keller et al . (1982) and Five days after protoplast culture, the cells Gleddie and Keller (1989). were transferred to Erlenmeyer flasks Protoplasts have served as recipient hosts for containing MS liquid medium and incubated at DNA transformation and are required in 120 rpm on a rotary shaker in the dark at 25 ± somatic hybridization by protoplasts fusion 2°C. After 10 days, every time, 5 ml of fresh (Stephen, l995). Protoplasts have been isolated medium was added to the culture medium. from various genotypes of petunia hybrida Star shaped microcalli developed within 15 (Izhar and Power, 1977), as well as from P. days of culture. After the development of inflata, P.violocea and P. axillaris (Dulieu et microcalli visible by naked eye, the cultures al ., 1983). Also, there are reports on isolation were transferred to the light. The plating and culture of protoplasts from leaves and efficiency was defined and measured as the Downloaded from jast.modares.ac.ir at 14:04 IRST on Tuesday October 5th 2021 suspension in specious such as Allium cepa ratio of cell number undergoing division to the (Karim and Adachi, 1997), Spathiphyllum and total cultured protoplast number. After one Anthurium (Duquenne et al ., 2007), Lotus month when the calli attained sizes of 0.5–1.0 corniculatus (Raikar et al ., 2008). mm in diameter, they were transferred to the To our knowledge, no report exists on semi-solidified MS medium at 23°C under protoplast technology in Lilium ledbourii fluorescent light (40 µmol per m 2 s-1) in a 16:8 (Baker) Boiss. In the present study, we report hour of day/night regime in the culture the induction of callus formation from cabinets. protoplasts in Lilium ledebourii for the first time. To optimize the preparation of protoplasts and cell division frequency, factors Experimental Designs, Data Collection, such as donor material, composition of and Analysis enzyme solutions, duration of incubation period, and culture system and the flowering In this study, three separate experiments of the regenerates through subsequent were done and each experiment was protoplast culturing were studied. repeated twice. In the first experiment, in order to optimize the medium for protoplast 1134 Plant Regeneration and Lilium ledebourii _______________________________________ growth and cell proliferation, the effect of completely randomized design with factorial various plant growth regulator combinations arrangement with three replications. The in MS medium (2 gr yeast extract (YE), 1 cultures were kept in light conditions of l6 and 2 mg L -1 2,4-D, 0.2 and 1 mg L -1 Kin) hours/day at 25°C. were tested as a suspension culture based on Cell density was estimated with a completely randomized design with factorial Nageotte hemocytometer. Results were arrangement and three replications. expressed as yield per gr FW for leaves or In the second experiment, to determine the calli. Length of bulbs, roots, and leaves were growth possibility of protoplast-derived cells measured by ruler. Callus mass was on the semi-solid medium, all of cells evaluated by naked eye. Data analyses were proliferated in suspension culture were sub- performed using SPSS (SPSS Inc. Version cultured on semi-solidified MS medium 19.0) software. Mean comparisons were supplemented with various combinations of done using Duncan’s multiple range test 2,4-D and kinetin (Kin) and yeast extract (1 (DMRT) at a probability level of 0.05. mg L -1 2,4-D and 0.2 mg L -1 Kin; 1 mg L -1 -1 -1 2,4-D and 1 mg L Kin; 1 mg L 2,4-D and RESULTS 0.2 mg L -1 Kin and 2 gr Yeast extract; 1 mg L-1 2,4-D and 1 mg L -1 Kin and 2 gr L -1 Yeast extract, 2 mg L -1 2,4-D and 0.2 mg L -1 First Experiment: Effect of Different Kin and 2 gr L -1 Yeast extract, 2 mg L -1 2,4- Hormones on Cell Growth and Deviation D and 1 mg L -1 Kin and 2 gr L -1 Yeast -1 -1 extract, 2 mg L 2,4-D and 0.2 mg L Kin). The results of ANOVA showed that After callus formation, callus mass were different concentrations of 2,4-D and counted. Kinetin significantly (P< 0.01) affected In the third experiment, after 26 days of proliferation of protoplast derived cells. callus proliferation, the developed calli in -1 Significant (P ≤ 0.01) interaction effects of suspension culture (1 mg L 2,4-D and 0.2 2,4-D×Kin, Yeast extract×Kin, Yeast mg L -1 Kin; 1 mg L -1 2,4-D and 1 mg L -1 -1 -1 extract×2,4-D and Yeast extract ×2,4-D×Kin Kin; 1 mg L 2,4-D and 0.2 mg L Kin and were found on cell proliferation (Table 1, -1 -1 2 gr L Yeast extract; 1 mg L 2,4-D and 1 Figure 1).