Zhi Ming Wei

We have developed an efficient procedure for plant regeneration of elite wheat cultivars using mature embryos. Firstly, we established the optimal combination of basal media, inoculation method and pretreatment method using biostatistical methods. The results indicated that the combination of MS medium and longitudinally bisected mature embryos showed the highest culture efficiency, whereas the pretreatment method had no significant effects on callus induction or plant regeneration. A 70% primary callus induction rate was achieved on MS medium containing 2 mg/l 2,4‐d for all tested cultivars. Primary calli were then transferred onto the subculture medium to initiate embryogenic calli. Supplementation of the subculture medium with the appropriate combination of phytohormones (2.0 mg/l 2,4‐d, 0.5 mg/l BA and 0.1 mg/l NAA) significantly enhanced embryogenic callus production. The addition of AgNO₃ (10 mg/l) in regeneration medium promoted plant regeneration, whereas CuSO₄ stimulated root formation. The use of this protocol achieved successful plant regeneration in eight tested cultivars. The culture efficiency ranged from 15.3% to 36.8%, suggesting this regeneration system may be an effective alternative for wheat genetic transformation.

Using petioles as explants, a cotton laccase cDNA (GaLA C1) was introduced into Populus alba var. pyramidalis by A. tumefaciens-mediated transformation. PCR and Southern blot analysis indicated that transgene was stably integrated into the genome of transformants. Enzyme assay showed that laccase activity was obviously increased in transformants. As compared with untransformed control, total lignin content in all tested transgenic lines was elevated in varying degrees (as highest as 21.5%). Histochemical staining of lignin further confirmed that overexpressing GaLA C1 could result in increased lignin content in transformants. Together, our data strongly suggested that GaLA C1 may participate in lignin synthesis and this is the first direct transgenic evidence for the involvement of plant laccases in lignification.

Four regeneration systems derived from cotyledon, hypocotyls, stem and needle explants have been established in the comparative researches of regeneration systems in Cunninghamia lanceolata Hook. A high frequency (93.7% +/- 0.45%) of adventitious buds were induced from cotyledons on DCR medium supplemented with 1 mg/L BA and 0.1 mg/L NAA, with a maximum mean number buds of 3.76 +/- 0.25. A higher frequency (96.2% +/- 0.35%) of adventitious buds were induced from hypocotyls on DCR medium supplemented with 2 mg/L BA and 0.2 mg/L NAA, with a maximum mean number buds of 17.4 +/- 0.18. A highest frequency (100%) of adventitious buds were induced from stem segments on DCR medium supplemented with 1 mg/L BA and 0.1 mg/L NAA, with a maximum mean number buds of 3.28 +/- 0.11. A low frequency (84.5% +/- 0.45%) of adventitious buds were induced from needles on DCR medium supplemented with 1.5 mg/L BA and 0.1 mg/L NAA, with a maximum mean number buds of 1.42 +/- 0.08. Buds were elongated on DCR medium supplemented with 0.2 mg/L BA and 0.02 mg/L NAA. After pre-treating shoots, best rooting result was produced on 1/2 DCR medium supplemented with 0.3 mg/L IBA.

Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans [Glycine max (L.) Merr.]. Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l⁻¹ N⁶-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l⁻¹ BAP and 0.2 mg l⁻¹ indolebutyric acid. Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials. Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h. Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8%. These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation.