Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation

Paz, Margie M.; Martı́nez, Juan Carlos; Kalvig, Andrea B.; Fonger, Tina M.; Wang, Kan

doi:10.1007/s00299-005-0048-7

Cited by 259 publications

(168 citation statements)

References 36 publications

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“…Direct organs are produced from the explant or callus during organogenesis, and this pathway has been most widely used for soybean regeneration. Various soybean explants that have been used to regenerate plants via organogenesis are stem node [31], primary leaf node [20], hypocotyl segments [22, 32], embryonic axes [33], half seed [34, 35], and cotyledonary node [14, 17, 36, 37]. However, the cotyledonary node remains the most preferred explants for soybean plant regeneration via organogenesis.…”

Section: Resultsmentioning

confidence: 99%

In VitroPlant Regeneration from Commercial Cultivars of Soybean

Raza

Singh

Bhalla

2017

BioMed Research International

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Soybean, a major legume crop, is the source of vegetable oil and protein. There is a need for transgenic approaches to breeding superior soybean varieties to meet future climate challenges. Efficient plant regeneration is a prerequisite for successful application of genetic transformation technology. Soybean cultivars are classified into different maturity groups based on photoperiod requirements. In this study, nine soybean varieties belonging to different maturity group were regenerated successfully from three different explants: half split hypocotyl, complete hypocotyl, and cotyledonary node. All the genotypes and explant types responded by producing adventitious shoots. Shoot induction potential ranged within 60–87%, 50–100%, and 75–100%, and regeneration rate ranged within 4.2–10, 2.7–4.2, and 2.6–10.5 shoots per explant using half split hypocotyl, complete hypocotyl, and cotyledonary explants, respectively, among all the tested genotypes. Bunya variety showed the best regeneration response using half split and complete hypocotyl explants and the PNR791 with cotyledonary node. The regenerated shoots were successfully rooted and acclimatized to glasshouse conditions. This study shows that commercial varieties of soybean are amenable to shoot regeneration with high regeneration frequencies and could be exploited for genetic transformation. Further, our results show no correlation between shoots regeneration capacity with the maturity grouping of the soybean cultivars tested.

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Section: Resultsmentioning

confidence: 99%

In VitroPlant Regeneration from Commercial Cultivars of Soybean

Raza

Singh

Bhalla

2017

BioMed Research International

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“…Since the CN method was applied to 28 diverse cultivars and/or genotypes of the soybean [17], improvements to this protocol have been suggested using half-seed explants of the Williams82 soybean cultivars (4.5%) which have been reported to increase the efficiency of transformation [19]. In our current study, we employed this adjusted CN method using half-seeds of the Korean soybean cultivar Kwangan in a three step treatment procedure (high concentration of Agrobacterium solution, sonication and vacuum) during the early stages of Agrobacterium inoculation.…”

Section: Discussionmentioning

confidence: 99%

“…Thus far, the most efficient method of soybean transformation has been found to be the Agrobacterium -mediated system based on the cotyledonary-node (CN) [16]–[18]. An improved CN method using half-seed explants has now been reported to reduce the time required for seed germination and precise shoot cutting [19].…”

Section: Introductionmentioning

confidence: 99%

Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression

Kim

et al. 2012

PLoS ONE

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The carotenoid biosynthetic pathway was genetically manipulated using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). The PAC gene was linked to either the β-conglycinin (β) or CaMV-35S (35S) promoter to generate β-PAC and 35S-PAC constructs, respectively. A total of 37 transgenic lines (19 for β-PAC and 18 for 35S-PAC) were obtained through Agrobacterium-mediated transformation using the modified half-seed method. The multi-copy insertion of the transgene was determined by genomic Southern blot analysis. Four lines for β-PAC were selected by visual inspection to confirm an orange endosperm, which was not found in the seeds of the 35S-PAC lines. The strong expression of PAC gene was detected in the seeds of the β-PAC lines and in the leaves of the 35S-PAC lines by RT-PCR and qRT-PCR analyses, suggesting that these two different promoters function distinctively. HPLC analysis of the seeds and leaves of the T2 generation plants revealed that the best line among the β-PAC transgenic seeds accumulated 146 µg/g of total carotenoids (approximately 62-fold higher than non-transgenic seeds), of which 112 µg/g (77%) was β-carotene. In contrast, the level and composition of the leaf carotenoids showed little difference between transgenic and non-transgenic soybean plants. We have therefore demonstrated the production of a high β-carotene soybean through the seed-specific overexpression of two carotenoid biosynthetic genes, Capsicum phytoene synthase and Pantoea carotene desaturase. This nutritional enhancement of soybean seeds through the elevation of the provitamin A content to produce biofortified food may have practical health benefits in the future in both humans and livestock.

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“…Regeneration through organogenesis was successfully obtained using cotyledonary nodes (Paz et al 2006), primary leaves (Wright et al 1987), shoot meristems (McCabe et al 1988) and mature seed-derived embryonic tips (Liu et al 2004). Though genetic transformation and subsequent plant regeneration via organogenesis is well established in this valuable crop (Liu et al 2004;Paz et al 2006;Hong et al 2007;Olhoft et al 2007), somatic embryogenesis is still highly preferred over organogenesis, as the transformants derived through embryogenesis are more uniform and the chances for the occurrence of variation among individual clones are lesser (Terzi and Lo Schiavo 1990;Osuga et al 1999). Furthermore, regeneration through somatic embryogenesis has certain advantages: (1) it is an efficient and high volume propagation system, hence less labour intensive, and (2) the embryogenic cultures developed from this system are pure and homogeneous, owing to their single cell origin (Jiménez 2001).…”

Section: Introductionmentioning

confidence: 99%

Regeneration of soybean (Glycine max L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

Loganathan

Maruthasalam

Shiu

et al. 2010

In Vitro Cell.Dev.Biol.-Plant

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We describe here a simple and efficient system of soybean (Glycine max L. Merrill) regeneration through direct somatic embryogenesis by using immature embryonic shoot tips (IEST) as explants. The cultivar Kaohsiung 10 (cv. K10) used in this study did not show embryogenic response either from mature seed-derived explants (cotyledon, embryonic tip, leaf, shoot and root) or immature cotyledons. However, it showed a high percentage (55.8%) of somatic embryo (SEm) formation from the IEST excised 2-3 wk after flowering, thus indicating the crucial roles of type and age of explants. The IEST put forth primary SEm after 2 mo of culturing on Murashige and Skoog (MS) medium supplemented with 6% sucrose, 164.8 A mu M 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM asparagine and 684 A mu M glutamine. Subsequently, secondary SEm were developed 1 mo after culturing on MS medium containing 123.6 A mu M 2,4-D and 3% sucrose. Cotyledonary embryos were induced on MS medium supplemented with 0.5% activated charcoal after 1 mo. The embryos were desiccated for 72-96 h on sterile Petri dishes and regenerated on hormone-free MS medium. Plantlets with well-developed shoots and roots were obtained within 5-6 mo of culturing of IEST. The SEm-derived plants were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration of soybean through somatic embryogenesis are discussed. To our knowledge, this is the first report to employ IEST as explants for successful direct somatic embryogenesis in soybean

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Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation

Cited by 259 publications

References 36 publications

In VitroPlant Regeneration from Commercial Cultivars of Soybean

In VitroPlant Regeneration from Commercial Cultivars of Soybean

Genetic Modification of the Soybean to Enhance the β-Carotene Content through Seed-Specific Expression

Regeneration of soybean (Glycine max L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

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