| US 7,368,631 B2 | ||
| Regeneration and genetic transformation of Acacia mangium | ||
| Deyu Xie, Singapore (Singapore); and Yan Hong, Singapore (Singapore) | ||
| Assigned to Temasek Life Sciences Laboratory Limited, Singapore (Singapore) | ||
| Filed on Dec. 09, 2004, as Appl. No. 11/7,194. | ||
| Application 11/007194 is a division of application No. 09/936612, granted, now 6,846,971, previously published as PCT/SG00/00010, filed on Jan. 19, 2000. | ||
| Prior Publication US 2005/0155116 A1, Jul. 14, 2005 | ||
| This patent is subject to a terminal disclaimer. | ||
| Int. Cl. C12N 5/04 (2006.01); C12N 5/10 (2006.01); C12N 15/82 (2006.01); C12N 15/84 (2006.01) | ||
| U.S. Cl. 800—294 [800/278; 435/410; 435/469; 435/430; 435/431; 435/420; 435/468] | 5 Claims |
| 1. A method of making transgenic Acacia mangium plants comprising:
a) culturing stem pieces of Acacia mangium cells on culture medium comprising basic MS medium supplemented with a) thidiazuron, b) indole-3-acetic acid, c) casein enzymatic
hydrolysate, d) L-ascorbic acid, e) L-glutamine, f) L-asparagine, g) L-proline, h) sucrose and i) phytagel or agar;
b) co-cultivating the stem pieces of step (a) with Agrobacterium in medium comprising basic MS medium supplemented with a) thidiazuron, b) indole-3-acetic acid, c) casein enzymatic hydrolysate,
d) L-ascorbic acid, e) L-glutamine, f) L-asparagine, g) L-proline, h) sucrose, i) acetosyringone and j) phytagel or agar to
form infected stem pieces;
c) culturing the infected stem pieces on medium comprising basic MS medium supplemented with a) thidiazuron, b) indole-3-acetic
acid, c) casein enzymatic hydrolysate, d) L-ascorbic acid, e) L-glutamine, f) L-asparagine, g) L-proline, h) sucrose, i) an
Agrobacterium eradicant, j) an antibiotic and k) phytagel or agar to induce and select transformed callus and adventitious bud formation
on the stem pieces;
d) culturing the stem pieces having callus and adventitious buds on a medium comprising basic MS medium supplemented with
a) thidiazuron, b) indole-3-acetic acid, c) casein enzymatic hydrolysate, d) L-ascorbic acid, e) L-glutamine, f) L-asparagine,
g) L-proline, h) sucrose, i) an antibiotic, j) gibberellic acid and k) phytagel or agar to promote adventitious bud elongation;
e) culturing the elongated adventitious buds on medium comprising basic MS medium supplemented with a) 6-benzylaminopurine,
b) gibberellic acid, c) casein enzymatic hydrolysate, d) L-ascorbic acid, e) L-glutamine, f) L-asparagine, g) L-proline, h)
sucrose, and i) phytagel or agar to promote pinnate leaf formation on the adventitious buds; and
f) culturing the adventitious buds with leaves on medium comprising ½ basic MS medium supplemented with a) α-naphthaleneacetic
acid, b) kinetin, c) casein enzymatic hydrolysate, d) L-ascorbic acid, e) L-glutamine, f) L-asparagine, g) L-proline, h) sucrose,
and i) phytagel to form rooted transgenic plantlets.
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