In vitro regeneration of commercial cultivars of subterranean clover

Heath, Lance C.; Chin, Shih-Foong; Spencer, Donald; Higgins, Thomas J.

doi:10.1007/bf00043938

Cited by 10 publications

(5 citation statements)

References 13 publications

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“…As with other plant species, the effectiveness of different types of cytokinins on somatic embryogenesis in legumes is species specific. For instance, different types of cytokinins were required for the optimal expression of somatic embryogenesis in different species of Medicago (Denchev et al, 1993 [32] ; Senaratna et al, 1995 [134] ; Trinh et al, 1998 [152] ; Nolan et al, 1989 [106] ; Li and Damarly, 1996 [84] ; Scarpa et al, 1993 [129] ) and Trifolium (Rybcsynski, 1997 [124] ; Radionenko et al, 1994 [119] ; Heath et al, 1993 [59] ). Though this differential cytokinin requirement could possibly stem from the genetic variability of the species studied, it might also be a reflection of the differential cytokinin sensitivity of diverse explant types used in the above investigations.…”

Section: Cytokininsmentioning

confidence: 99%

Somatic Embryogenesis in Leguminous Plants

Pugalendhi¹,

Taji

2000

Plant Biology

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This review examines recent advances in the induction and development of somatic embryos in leguminous plants. Emphasis has been given to identify the current trends and successful strategies for the establishment of somatic embryogenic systems, particularly in the economically important species. It appears that, in legumes, somatic embryogenesis can be realized relatively easily especially in young meristematic tissues such as immature embryos and developing leaves. In the majority of the species examined, chlorophenoxyacetic acids remained the most active inductive compounds; however, the new generation growth regulators such as thidiazuron are emerging as successful alternatives for high-frequency direct regeneration of somatic embryos, even from well differentiated explant tissues. Low-frequency embryo production, poor germination and conversion of somatic embryos into plantlets and somaclonal variation are the major impediments limiting the utility of somatic embryogenesis for biotechnological applications in legumes. These limitations, however, may be considerably reduced in the near future, as more newly developed growth regulators with specific morphogenic targets become available for experimentation. From the published data, it is apparent that more effort should be given to develop repetitive embryogenic systems with high frequency of germination and regeneration, since such systems will find immediate application in mass propagation and other crop improvement programmes. As our understanding of various morphogenic processes, including growth and differentiation of zygotic embryos, is fast expanding, it is conceivable that development of highly efficient somatic embryogenic systems with practical application can be anticipated, at least for the important leguminous crops, in the foreseeable future.

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Section: Cytokininsmentioning

confidence: 99%

Somatic Embryogenesis in Leguminous Plants

Pugalendhi¹,

Taji

2000

Plant Biology

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“…The regeneration system for subterranean clover reported earlier (Heath et al, 1993) has been modified by replacing the phytohormone benzylaminopurine with TDZ because the latter was found consistently to induce a higher number of adventitious shoots from hypocotyl explants, thus enabling the production of more shoots in selection medium. Adventitious shoots were formed on the periphery of hypocotyl segments grown on the regeneration medium (Fig.…”

Section: Development Of Transformation and Regeneration Proceduresmentioning

confidence: 99%

“…Because of the importance of subterranean clover in Australian agriculture, we set out to develop a gene transfer system for this clover so that useful genes can be introduced by genetic engineering techniques. A regeneration system for subterranean clover has recently been developed (Heath et al, 1993) using hypocotyl tissues from germinating seeds. We have adapted this procedure and used it in conjunction with a disarmed strain of Agrobacterium tumefaciens to deliver four genes to subterranean clover.…”

Section: Australiamentioning

confidence: 99%

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.)

et al. 1994

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We have developed a rapid and reproducible transformation system for subterranean clover (Trifolium subterraneum 1.) using Agrobacterium tumefaciens-mediated gene delivery. Hypocotyl segments from seeds that had been allowed to imbibe were used as explants, and regeneration was achieved via organogenesis. Clucose and acetosyringone were required in the co-cultivation medium for efficient gene transfer. DNA constructs containing four genes encoding the enzymes phosphinothricin acetyl transferase, 8-glucuronidase (GUS), neomycin phosphotransferase, and an a-amylase inhibitor were used to transform subterranean clover. lransgenic shoots were selected on a medium containing 50 mg/L of phosphinothricin. Four commercial cultivars of subterranean clover (representing all three subspecies) have been successfully transformed. Southern analysis revealed the integration of 1-DNA into the subterranean clover genome. l h e expression of the introduced genes has been confirmed by enzyme assays and northern blot analyses. Transformed plants grown in the glasshouse showed resistance to the herbicide Basta at applications equal to or higher than rates recommended for killing subterranean clover in field conditions. In plants grown from the selfed seeds of the primary transformants, the newly acquired gene encoding CUS segregated as a dominant Mendelian trait. ~ Subterranean clover (Trifolium subterraneum L., subclover) is native to the Mediterranean regions and is grown as a pasture legume mainly in Australia and the countries around the Mediterranean. To a lesser extent, it is also grown in the Americas, Africa, New Zealand, and Japan (Johnstone and McLean, 1987). In Australia, subterranean clover is the major pasture legume and is grown on more than 16 million ha of mainly acidic and infertile lands. As a pasture crop and because of its ability to fix nitrogen, subterranean clover is a major contributor to the productivity of the meat, wool, dairy, and wheat industries in Australia (Johnstone and McLean, 1987). It also has the potential to be grown in other temperate regions of the world. A number of grain and pasture legumes are now amenable to gene transfer by genetic engineering using the Agrobacte-rium-mediated gene delivery system. These include soybean (Hinchee et al.

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“…We also suggest that future studies should test the effect of cefotaxime on root formation. The efficiency of root formation using 1.2 µM IAA was found to be 16.6% [24], which might be improved with the addition of cefotaxime in the rooting medium. As an antagonist effect, 500 mg L −1 cefotaxime tended to reduce the mean explant fresh weight in relation to 200 mg L −1 , but this did not reach significance (p < 0.05).…”

Section: Cefotaxime Positive Effect On Shoot Regenerationmentioning

confidence: 95%

“…Four rooting media were tested with different combinations of hormones, all of them containing half-strength MS salts and MS vitamins plus 15 g L −1 of sucrose: (i) 1.2 µM indole-3-butyric acid (IBA) [10] (PhytoTechnology Laboratories ® , Germany), (ii) 3 mg L −1 IBA [11], (iii) 1.2 µM indole-3-acetic acid (IAA) (Sigma-Aldrich ® , Munich, Germany) [24], and (iv) 5 µM IAA and 1 µM Kinetin (Sigma-Aldrich ® , USA) [30]. The presence of roots was scored after 2 months of culture in rooting medium.…”

Section: Explant Preparation and In Vitro Culturementioning

confidence: 99%

An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.)

Rojo

Seth

Erskine

et al. 2021

IJMS

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Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L−1 hygromycin, 100 mg L−1 kanamycin and 200 mg L−1 cefotaxime. Additionally, 200 mg L−1 cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.

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In vitro regeneration of commercial cultivars of subterranean clover

Cited by 10 publications

References 13 publications

Somatic Embryogenesis in Leguminous Plants

Somatic Embryogenesis in Leguminous Plants

Agrobacterium-Mediated Transformation of Subterranean Clover (Trifolium subterraneum L.)

An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.)

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