A Simple and Efficient Protocol for Plant Regeneration and Genetic Transformation of Tomato cv. Micro-Tom from Leaf Explants

Abstract

<jats:p>A simplified protocol to obtain transgenic tomato plants was established. The effects of culture media composition and <jats:italic>Agrobacterium</jats:italic> concentration were evaluated. The highest shoot-forming capacity index (5.6) was observed when leaf explants were cultured for 6 weeks with 2 mg·L<jats:sup>−1</jats:sup> zeatin, 0.1 mg·L<jats:sup>−1</jats:sup> indoleacetic acid, and 300 mg·L<jats:sup>−1</jats:sup> timentin. Shoot elongation and root formation were performed in one step on growth regulator-free media. The highest percentage (82%) of fully developed plantlets was obtained when shoots were cultured for 4 weeks with 0.5× Murashige and Skoog (MS) media and 15 g·L<jats:sup>−1</jats:sup> sucrose. A 100% of plant survival rate was observed after 4 weeks of being transplanted to ex vitro conditions followed by fruit production (15 fruits/plant) after 2 more weeks. Transient expression of β-glucuronidase was visualized in 100% of the leaf explants infected with <jats:italic>Agrobacterium</jats:italic> at an OD<jats:sub>600</jats:sub> = 0.5 and cocultured for 48 h with 2 mg·L<jats:sup>−1</jats:sup> benzylaminopurine, 0.1 mg·L<jats:sup>−1</jats:sup> naphthaleneacetic acid, and 100 μM acetosyringone. Stable transformation was confirmed by histochemical glucuronidase assay and polymerase chain reaction (PCR) analysis with a total efficiency of 19.1%. The complete protocol, from shoot induction to fruit production of soil-adapted transgenic plants can be accomplished in only 4 months, and it seems to be very useful for both micropropagation and genetic transformation purposes.</jats:p>

DNA methylation dynamics of sperm cell lineage development in tomato

“…Several evidence lines have shown that decreased DNA methylation might promote the microspore's embryogenesis initiation in Brassica and triticale (Solís et al ., 2012, 2015; Nowicka et al ., 2019). Tomato microspores showed similar DNA methylome with leaves, and they both can be induced for plant regeneration (Segui‐Simarro and Nuez, 2007; Cruz‐Mendívil et al ., 2011). However, to our knowledge, no evidence showed the feasibility of plant regeneration from the daughter cells of asymmetric division.…”

Genetic Improvement of Tomato (Solanum lycopersicum) with AtDREB1A Gene for Cold Stress Tolerance using Optimized Agrobacterium-mediated Transformation System

“…Micro-Tom and concluded that Agrobacterium suspension (OD600 nm = 0.5) along with other optimized parameters yielded the maximum TE (5.1%) culturing cotyledonary explants. Similarly, Cruz-Mendivil et al (2011) conducted transformation study and inferred that leaf explants co-infected with bacterial density (OD600 nm = 0.5) produced the maximum TE (19.1%) in tomato cv. Micro-Tom.…”

Mitigating Root Knot Nematode Propagation on Transgenic Tobacco via &lt;i&gt;in Planta&lt;/i&gt; Hairpin RNA Expression of &lt;i&gt;Meloidogyne incognita&lt;/i&gt;—Specific PolA1 Sequence

“…One-internode stem cuttings of 3 selected transgenic (T1, T4 and T7) and WT tobacco lines were rooted in 1/2 MS medium containing 1% sucrose and 0.8% agar for two weeks. Rooted plants were acclimatized on sterile vermiculite [17], transferred to pots containing 3000 ml of sterile commercial garden soil and allowed further acclimatization in the glasshouse for 12 days. They were then individually infected with approximately 10,000 Meloidogyne incognita eggs of same batch via 3 holes made around each plant root system.…”