Optimization of a plant regeneration and genetic transformation protocol for Eucalyptusclonal genotypes
© Deepika et al; licensee BioMed Central Ltd. 2011
Published: 13 September 2011
Agrobacterium mediated gene transfer technology offers the potential to introduce novel, high-value traits into selected, elite tree genotypes. Significant progress has been made in the regeneration and genetic transformation of Eucalyptus trees, but transformation efficiency has generally been low for eucalypts , especially for elite clonal genotypes. As a preliminary step towards the regeneration of explants derived from clonal material and the production of transgenic plants from commercially important Eucalyptus genotypes, there is a need to identify genotypes exhibiting high regeneration capacity. In this ongoing study, we are comparing eight Eucalyptus genotypes for callus induction and shoot regeneration potential. Browning of callus tissue and surrounding culture media is a common obstacle limiting regeneration of shoots in eucalypts. Hence, a second aim is to minimize the oxidation of phenolic compounds released from the wounded tissue and improve shoot regeneration rates.
Shoot buds collected from potted ramets of eight Eucalyptus clones (Sappi and Mondi, South Africa) were established under in vitro conditionson MS  basal medium containing BAP (Benzyl adenine purine). The established clones consisted of four E. grandis (G1, G2, G3 and G4), one E. grandis x E. nitens (GN1), two E. grandis x E. camaldulensis (GC1 and GC2) and one E. grandis x E. urophylla (GU1) genotype. Leaf explants were excised from in vitro shoot cultures and cultured on MS basal medium containing BAP, NAA (Naphthalene acetic acid) and TDZ (Thidiazuron). Browning and necrosis of the callus as well as surrounding culture media was observed in all cultures and PVP (polyvinylpyrrolidone) was therefore added to the media to reduce oxidation.
A standardised regeneration protocol was used for the optimization of Agrobacterium mediated genetic transformation of selected Eucalyptus clones. The Agrobacterium tumefaciens strain AGL1 containing pMDC162 with the uidA gene (β-glucuronidase; GUS) and hpt (hygromycin phosphotransferase) gene was used for transformation. To minimize extensive exudation of phenolic compounds from the wounded explants, the leaves were subjected to different wounding methods. Intact leaves were excised from in vitro shoot cultures and preconditioned for 24 hrs on callus induction medium followed by wounding with a surgical blade prior to Agrobacterium infection. The leaves were wounded in two different ways: (a) removing the edges of the leaf, making 8-10 small wounds and cutting the leaf transversally into two equal halves and (b) making 5-6 small wounds in the intact leaf without damaging the rest of the leaf. Alternatively, the leaves were wounded prior to 24 h preconditioning using the first wounding method (a). The explants were immersed in a bacterial solution (A600 = 0.8) for 1 hr followed by blotting with sterile filter paper. The explants were co-cultivated for 24 hrs and stained for GUS activity after two days to determine the percentage of explants showing transient GUS expression.
Results and discussion
Different wounding methods did not have a significant effect on transient GUS expression in leaf explants. However, good GUS activity was detected in the transformed callus induced from the leaf explants wounded prior to preconditioning.
We have achieved efficient and rapid callus induction, as well as shoot regeneration for selected Eucalyptus clones. The information presented here forms the basis for ongoing optimization of a protocol for the generation of transgenic plants of clonal Eucalyptus genotypes.
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