Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium

Wakasa, Yuhya; Kasai, Atsushi; Yamazaki, Muneo; Tabei, Yutaka; Tsuyama, Mutsuo; Igarashi, Toshiya; Okazaki, Tomokazu; Yamamoto, Kayo; Fujihara, Hideki; Kanno, Ayumi; Noro, Osamu; Harada, Takeo; Akada, Shinji

doi:10.1007/s00299-020-02572-6

Cited by 3 publications

(5 citation statements)

References 25 publications

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“…The most efficient factors for microtuber induction are increased sucrose concentrations (5–8%) [ 15 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ] and the addition of cytokinins. Wakasa et al [ 20 ] analyzed gene expression using microtubers and were found to be an excellent tool for the analysis of potato genes that are expressed in normal tubers. In our protocol, potato microtuberization was achieved in osmotic stress conditions using sucrose (8%) and high content of gelrite (6 g/L) compared to non-osmotic stress (MS, sucrose 1%, gelrite 3 g/L) MR1-G3-2iP after four weeks of incubation.…”

Section: Discussionmentioning

confidence: 99%

“…The induction of microtubers under in vitro conditions was first described by Baker [ 15 ]; Mes and Menge [ 16 ], using high content of sucrose in the medium, as plant pathology tool. An extensive number of articles since then have been published for basic and practical research [ 17 , 18 , 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

“…An extensive number of articles since then have been published for basic and practical research [17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

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Gene Expression Analysis of Microtubers of Potato Solanum tuberosum L. Induced in Cytokinin Containing Medium and Osmotic Stress

Herrera-Isidrón

Valencia-Lozano

Rosiles-Loeza

et al. 2021

Plants

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Potato microtuber productions through in vitro techniques are ideal propagules for producing high quality seed potatoes. Microtuber development is influenced by several factors, i.e., high content sucrose and cytokinins are among them. To understand a molecular mechanism of microtuberization using osmotic stress and cytokinin signaling will help us to elucidate this process. We demonstrate in this work a rapid and efficient protocol for microtuber development and gene expression analysis. Medium with high content of sucrose and gelrite supplemented with 2iP as cytokinin under darkness condition produced the higher quantity and quality of microtubers. Gene expression analysis of genes involved in the two-component signaling system (StHK1), cytokinin signaling, (StHK3, StHP4, StRR1) homeodomains (WUSCHEL, POTH1, BEL5), auxin signaling, ARF5, carbon metabolism (TPI, TIM), protein synthesis, NAC5 and a morphogenetic regulator of tuberization (POTH15) was performed by qPCR real time. Differential gene expression was observed during microtuber development. Gene regulation of two component and cytokinin signaling is taking place during this developmental process, yielding more microtubers. Further analysis of each component is required to elucidate it.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Gene Expression Analysis of Microtubers of Potato Solanum tuberosum L. Induced in Cytokinin Containing Medium and Osmotic Stress

Herrera-Isidrón

Valencia-Lozano

Rosiles-Loeza

et al. 2021

Plants

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“…In vitro microtubers were induced using the method of Wakasa et al (2020) with modifications as follows. Apical shoot segments (1 cm length) containing a single leaf and one to two axillary buds were obtained from 3-week-old in vitro virus-free and virus-infected stock plants.…”

Section: Methodsmentioning

confidence: 99%

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies

et al. 2022

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Certain viruses dramatically affect yield and quality of potatoes and have proved difficult to eradicate with current approaches. Here, we describe a reliable and efficient virus eradication method that is high throughput and more efficacious at producing virus-free potato plants than current reported methods. Thermotherapy, chemotherapy, and cryotherapy treatments were tested alone and in combination for ability to eradicate single and mixed Potato virus S (PVS), Potato virus A (PVA), and Potato virus M (PVM) infections from three potato cultivars. Chemotherapy treatments were undertaken on in vitro shoot segments for four weeks in culture medium supplemented with 100 mg L−1 ribavirin. Thermotherapy on in vitro shoot segments was applied for two weeks at 40°C (day) and 28°C (night) with a 16 h photoperiod. Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture followed by exposure to PVS2 either without or with liquid nitrogen (LN, cryotherapy) treatment. The virus status of control and recovered plants following therapies was assessed in post-regeneration culture after 3 months and then retested in plants after they had been growing in a greenhouse for a further 3 months. Microtuber production was investigated using in vitro virus-free and virus-infected segments. We found that thermotherapy and cryotherapy (60 min PVS2 + LN) used alone were not effective in virus eradication, while chemotherapy was better but with variable efficacy (20–100%). The most effective result (70–100% virus eradication) was obtained by combining chemotherapy with cryotherapy, or by consecutive chemotherapy, combined chemotherapy and thermotherapy, then cryotherapy treatments irrespective of cultivar. Regrowth following the two best virus eradication treatments was similar ranging from 8.6 to 29% across the three cultivars. The importance of virus removal on yield was reflected in “Dunluce” free of PVS having higher numbers of microtubers and in “V500’ free of PVS and PVA having a greater proportion of microtubers > 5 mm. Our improved procedure has potential for producing virus-free planting material for the potato industry. It could also underpin the global exchange of virus-free germplasm for conservation and breeding programs.

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“…This fraction (10 µl) was used for 7.5% SDS-polyacrylamide gel electrophoresis and subjected to western blot analysis using antiserum raised against rice BE1, which was prepared from developing rice seeds (Kawasaki et al 1996). SDS-PAGE and western blot analysis were performed according to Wakasa et al 2020. For this analysis, a 5,000-fold dilution of the antiserum was used.…”

Section: Protein Extraction Sds-page and Western Blot Analysismentioning

confidence: 99%

Creation of a potato mutant lacking the starch branching enzyme gene <i>StSBE3</i> that was generated by genome editing using the CRISPR/dMac3-Cas9 system

Takeuchi

Ohnuma

Teramura

et al. 2021

Plant Biotechnology

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The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.

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Rapid analysis of GBSS1 and Vinv genes expressed in potato tubers using microtubers produced in liquid culture medium

Cited by 3 publications

References 25 publications

Gene Expression Analysis of Microtubers of Potato Solanum tuberosum L. Induced in Cytokinin Containing Medium and Osmotic Stress

Gene Expression Analysis of Microtubers of Potato Solanum tuberosum L. Induced in Cytokinin Containing Medium and Osmotic Stress

Eradication of Potato Virus S, Potato Virus A, and Potato Virus M From Infected in vitro-Grown Potato Shoots Using in vitro Therapies

Creation of a potato mutant lacking the starch branching enzyme gene <i>StSBE3</i> that was generated by genome editing using the CRISPR/dMac3-Cas9 system

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