Research Article: Efficient and reproducible somatic embryogenesis and micropropagation in tomato via novel structures – Rhizoid Tubers

Date Published: May 22, 2019

Publisher: Public Library of Science

Author(s): Wajeeha Saeed, Saadia Naseem, Daniyal Gohar, Zahid Ali, Allah Bakhsh.


A dual in vitro regeneration system consisting of indirect organogenesis and somatic embryogenesis (SE), applicable to several varieties of tomato—Solanum lycopersicum (cv. Riogrande, cv. Roma, hybrid 17905 and model cv. M82) has been established. This system is both improved and highly reproducible compared to current methods. Callus initiation, plant regeneration and SE was developed for one-week-old cotyledon explants. Indirect organogenesis via callus induction (CI) was developed for all four varieties of tomato used in this study. One-week-old tomato seedlings were used as a source of cotyledon and hypocotyl segments as explants. The explants were subsequently cultured on Murashige and Skoog (MS) medium supplemented with different combination and concentrations of plant growth regulators (PGRs). Substantial trends in regeneration and propagation response were observed among the varieties and treatments. For commercial varieties cvs. Riogrande and Roma, maximum CI was observed at 2 weeks in CIMT9 (0.5 mg/L NAA, 1 mg/L BAP) and CIMT12 (2 mg/L IAA, 2 mg/L NAA, 2 mg/L BAP, 4 mg/L KIN). However, cv. M82 responded after 4 weeks to a combination of treatments CIMT9 (0.5 mg/L NAA + 1 mg/L BAP) and CIMT13 (2 mg/L IAA + 2 mg/L NAA + 2 mg/L BAP + 4 mg/L ZEA) for the production of calli. Subsequent shoot and root organogenesis were optimized for all four varieties. Cv. Riogrande, exhibited fastidious in vitro regeneration potential and selected for induction of somatic embryos via SE involving novel structure: rhizoid tubers (RTBs). Numerous fine hair like rhizoids (~23/explants) were first developed from cotyledon and hypocotyl explants cultured on MS medium supplemented with 0.5 or 2 mg/L NAA at pH 4.0 in dark conditions. Further incubation of each rhizoid under light conditions on MS media supplemented with 5 mg/L TDZ or BAP at pH 4.0 led to the formation of a novel structure—rhizoid Tubers (RTBs). Thus, as evident from histology, SE in Riogrande tomato species requires a medium with pH of (4.0) and higher concentration of cytokinins (BAP/TDZ) to form on average 40–45 RTBs from both explants. Histological and morphological studies revealed that RTBs develop through different stages of embryogenesis to multiple plantlets, on MS medium with 5 mg/L TDZ/BAP at normal pH (5.8). The results obtained indicated that the induced somatic embryos of tomato with lower pH are a more efficient mode of propagation than the organogenesis with or without callus formation. The RTBs led to a complete plantlets regeneration in 45 days compared to indirect organogenesis at 60 days.

Partial Text

Tomato (Solanum lycopersicum) is one of the most important edible perennial crop from the Solanaceae family. Relatively small genome, a diverse germplasm, and the availability of a suitable transformation system has made tomato an ideal model system for the improvement of other dicotyledonous plants [1,2]. Approximately 177 million tons of tomatoes were produced in 2016 worldwide [3]. China is playing a leading role in tomato production followed by India and the United States. Within Europe, Italy and Spain are the major producers–contributing 2/3 of the total 18 million tons of tomatoes producedin 2016 [4]. In 2014, Pakistan produced approximately 0.6 million tons of tomatoes on 62,930 hectares of land [5]. Regardless of favorable environment and available infrastructure for mass production of tomatoes, marketable yields are decreasing due to weather conditions (continuous rainy seasons and floods) and pre/post-harvest losses [6]. Being a highly perishable horticulture commodity, tomatoes are prone to post-harvest losses. These losses, however, are more prominent in developing countries where loss index can reach up to 50% annually [7,8]. Several other biotic and abiotic factors reduce the global production of tomatoes. To overcome these barriers, there has been an increased demand for the introduction of qualitative traits into commercially important cultivars of tomato in order to increase their productivity, nutritional value, and shelf life [1,9,10]. Sustainable tomato cultivation along with conventional breeding practices requires an effective regeneration system, which can exploit tissue and cell cultures for genetic amelioration with desired traits. Such system could be of potential use in somatic embryogenesis (SE), germplasm conservation and development of elite transgenic plants.

In the present study, various physical and chemical factors were optimized for somatic embryogenesis and in vitro regeneration of commercially important and model tomato cultivar(s).

Conventional breeding management augmented with sustainable in vitro cell cultures can be exploited for the production of genetically engineered plants. The development of this system will be of great value in germplasm conservation, somatic embryogenesis, and the multiplication of in vitro grown seedlings.

Considering the commercial and economic importance of Rio tomatoes, the regeneration system via SE involving newly identified structures (RTBs) in tomato will enhance the introduction of economically important traits in transgenic plants as it can yield multiple plantlets of tomato from single explant in 45 days. In many plant species induction of SE and recovery of viable plantlets is not frequently achievable. Nonetheless, the connotation SE has gained importance in clonal propagation as a useful tool to be used instead of conventional micro-propagation. While secondary embryos serve as excellent choice of explant for gene transfer technology. In addition, the RTBs and SE regeneration system could be used to overcome recalcitrance phenomenon in tissue culture and transformation of various other varieties of tomato. The results obtained are relevant for speed up of breeding program by production of large scale true to type plants of tomato lines with desired characteristics. Such system can be applied to predict the embryogenic competence of particular lines and their subsequent use for transformation and large-scale propagation. The RTBs will be available as a juvenile source of explant with high capacity to yield plantlets. Further, dedicated studies can confirm ubiquity of RTBs as secondary embryos and in vitro regeneration in other plant species as well.




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