Optimization of Isolation and Culture of Protoplasts in Alfalfa (<i>Medicago sativa</i>) Cultivar Regen-SY

Abstract

Alfalfa (Medicago sativa) is an important forage crop belonging to the Fabaceae family. It is cultivated across the world for fodder and originated in Asia. Alfalfa cultivar Regen-SY was used in this study which is a hybrid of first-generation self-parents from Regen-S (M. sativa) and Regen-Y (Medicago falcata) research cultivars. The main objective of the study was to optimize conditions for the isolation and liquid culture of alfalfa Regen-SY protoplasts. Several factors like enzyme combination, incubation time, plant age, centrifugation speed and shaker speed affecting protoplast isolation and culture were optimized in the study. The yield and viability of the protoplasts was determined by using hemocytometer and Fluorescein diacetate (FDA) staining respectively. Results showed that factors like enzyme combination, incubation time, plant age, centrifugation speed and Mannitol concentration significantly (p ≤ 0.05) affect protoplast yield and viability whereas shaker speed didn't result in any significant difference in the yield and viability of protoplasts. Using optimum conditions protoplasts were cultured in the liquid medium and microcalli formation was achieved after five weeks of the culture. The protocol established in this study will assist researchers in the isolation and culture of protoplasts in alfalfa and will accelerate the research processes like protoplast fusion and genetic engineering.

Generation of Yeast Protoplasts by Lytic Actions of Iron Oxide Magnetic Nanoparticles

“…16 Bands were observed at the expected size for the targeted DNA, i.e., around 500 bp (Figure 8), whereas no product was observed with untransformed/empty cells serving as a negative control. It is of note that, to achieve sufficiently high efficiency for practical applications, there is still need to optimize conditions (such as osmotic stabilizers 35 ) for preparation and transformation of the protoplasts in the future studies. These results demonstrated the great potential of the as-prepared protoplasts from S. cerevisiae yeast cells for genetic manipulation.…”

Protoplast: A Valuable Toolbox to Investigate Plant Stress Perception and Response

“…Indeed, protoplast isolation procedures and the culture conditions that follow, may induce cell stress or damage ( Neelakandan and Wang, 2012 ), which should be avoided if one wants to study the effect of biotic and abiotic stresses. One of the most referenced dyes is FDA (fluorescein diacetate), which highlights living cells ( Bertini et al, 2019 ; Sangra et al, 2019 ; Qiu et al, 2020 ) or Evan’s blue, which highlights dead ones ( Kollárová et al, 2019 ). Other fluorochromes can be used, such as PI (propidium iodide), DAPI (4'6-diamidino-2-phenylindole, dichloride), and 7-AAD (7-amino-actinomycin D) that do not cross intact PMs.…”

“…Nonetheless, the single cell level allows for specific, rapid, and high-throughput analysis along with time-course experiments. In addition, even though protoplasts isolation and maintenance require specific conditions that can eventually cause stress, these techniques are improving for a wide range of species or organs ( Sangra et al, 2019 ; Shan et al, 2019 ; Davis et al, 2020 ). Furthermore, their formation is still deemed necessary to bypass time-consuming plant culture and whole plant transformation, especially for recalcitrant species or plants with a long reproductive cycle ( Du and Bao, 2005 ).…”

“…The first protoplast isolations were developed in bacteria ( Weibull, 1953 ) and fungi ( Eddy and Williamson, 1957 ; Barbara and Bonner, 1959 ), before being transposed to plants ( Cocking, 1960 ). They are usually obtained from enzymatic digestion of leaf and root tissues or even from cultured cells of a wide variety of species ( Fowke et al, 1983 ; Yoo et al, 2007 ; Lin et al, 2018 ; Sangra et al, 2019 ; Zhao et al, 2019 ; Cheng and Nakata, 2020 ). With transformation methods already developed and microscopy techniques fast expending, the protoplast system could ultimately be considered as convenient screening platform to better target future whole plant analyses ( Li et al, 2014 ).…”

Embryogenic Callus Differentiation in Short-Term Callus Derived from Leaf Explants of Alfalfa Cultivars

“…Callus is a primary tool in order to induce embryogenic callus and somatic embryos in crops. Callus of alfalfa or any explants is essentially a more and fewer undifferentiation structure which generally develops on wounds of exogenous stimulates (Dos Santos et al, 1983;Fehér, 2015;Sangra et al, 2019). Somatic embryo induction is linked to the ability of embryogenic callus formation.…”