Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp.

Mohamed A A Ahmed,Emmanouil D Pratsinakis,Meiling Lyu,Panagiotis Madesis,Binghua Wu,Wei Wang,Hongliang Zhang,Miao Miao,Yuan Yuan,Ahmed F Yousef,Junaid Iftikhar

doi:10.3390/agriculture11080699

Abstract

Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

Highlights

Calli could be induced from stem explants with inconstant efficacy, responding to a large range of combinations, including 6-benzylaminopurine (2 mg/L 6-BA) and 1-naphthaleneacetic acid (0.2 mg/L naphthylacetic acetic acid (NAA)) on modified woody plant medium (WPM) basal medium
The most effective medium for callus induction from stem segments was WPM or/and Murashinge and Skoog (MS) supplied with 2 mg/L 6-BA
Discussion dependency on shoot cultures as a general supplier of tissue has the distinctive benefit of eliminating the difficulties related to seasonal growth, dormancy and extended in vitro establishment times that must be faced any time when new explants of woody species are re-isolated for any use

Summary

Introduction

Jasminum sambac (L.) Ait., an evergreen shrub, is widely cultivated as an ornamental plant in the southern and eastern parts of India. Its flowers provide important raw materials for scented tea and essential oils. Medicinal, and ornamental value of J. sambac, the major problem encountered in its cultivation is the lack of intraspecies genetic variation [1]. Jasmine is considered a recalcitrant species regarding tissue culture and regeneration [2]. Overcoming difficulties in J. sambac regeneration could be a critical step for future studies and for genetic diversity through callus formation [2]

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Discussion

Conclusion