# Patent

## Title
Regeneration and genetic transformation of Acacia mangium

## Abstract

The present invention is directed to a method of Acacia mangium regeneration through organogenesis and a method of genetic transformation of Acacia mangium. The method of regeneration comprises inducing callus from different parts of seedlings and vegetatively micropropagated plantlets as explants; adventitious bud induction followed by pinnate leaf and bud elongation and eventually, elongated shoots were induced to root. Based on the regeneration system, a marker gene GUS under cauliflower mosaic virus promoter was introduced to Acacia mangium via Agrobacterium infection. GUS staining in the regenerated plants and Southern blot hybridization prove the incorporation of the foreign gene into the host genome and expression of the foreign gene.

## Background
BACKGROUND OF THE INVENTION
Forests are very important to the world economy and for maintaining and preserving our ecosystem. Forest trees have a wide range of commercial uses (timber for construction, raw material for paper and pulp production, and as an energy source). The global demand for wood products (mostly for paper and pulp and firewood in developing countries) has been increasing year by year when the natural forests are in short supply. Re-forestation is the solution to meeting such increasing demand. Usually, the fast-growing, widely adapted tree species are chosen for re-forestation. Most tree-improvement programs are based on the management of genetic resources, including the selection of superior clones from existing forests, the conservation of genetic variability, partially controlled propagation and classical breeding for desired traits. In spite of the fact that it usually takes several generations to breed, this traditional breeding has been successful in getting elite trees with fast and uniform growth. However, many other traits such as disease and insect resistance, different lignin composition and content are difficult to acquire mainly due to high heterozygosity in tree species and big segregation population. Moreover, the gene(s) conferring certain phenotypes like disease resistance may not be in the gene pool at all. On the other hand, molecular breeding based on genetic transformation of tree species offers the possibility to introduce a particular phenotype without affecting the genetic background of a cultivar. Genetic transformation in Populus species and Eucalyptus species enabled some success in modification of lignin content (rzfira et al., 1998; Robinson, 1999). The precondition of molecular breeding of forest tree species is the availability of a reliable and reproducible genetic transformation method, which in turn relies on a system of regeneration of one whole plant from a single cell.
Genus Acacia comprises about 1200 tropical and subtropical tree species. It belongs to the family Mimosaceae. Acacia mangium is a multipurpose, fast growing and nitrogen fixing elite tropical legume tree. An adult tree is up to 30-meters tall and its bole (trunk) is often straight to over half the total height. The true leaf of a seedling is a fem-like pinnate leaf. The first pinnate leaf of a seedling has 6 or 8 leaflets, and then the bipinnate leaf develops from the second pinnate leaf on a seedling. Usually when the young seedling grows up to 8-12 bipinnate leaves, the petiole is dilated into a phyllode, while the leaflets abort completely and the true leaf disappears from the young seedling. Phyllodes are flattened leaf stems which look and act like the ordinary leaves of other plants. Branchlets, phyllodes and petioles are glabrous or slightly scurfy. Phyllodes are 5-10 cm broad, 24 times as long as broad, dark green, and are chartaceous when dry. The phyllodes have 3-4 longitudinal main nerves which join on the dorsal margin at the base of the phyllode. Secondary nerves are fine and inconspicuous.
Flowers are in loose spikes to 10 cm long and are solitary or paired in the upper axils. Flowers are pentamerous and the calyx is 0.6-0.8 mm long with short obtuse lobes. The corolla is twice as long as the calyx. Pods are linear, glabrous, 3-5 mm broad, about 7.5 cm long when green, woody, coiled and brackish-brown when mature, and depressed between the seeds. Seeds are lustrous, black, ellipsoid, ovate or oblong, 3.5×2.5 mm, with the orangish funicle forming a fleshy aril beneath the seed (Duke, 1984). Due to A. mangium fast growth, tolerance of infertile soil and high quality fibre, it has been increasingly used for reforestation plantation and soil rehabilitation in degraded soil. Its plantation has been studied for many years in many countries or regions, especially in tropical regions and subtropical regions, such as Australian, Indonesia, Malaysia, India, Thailand, Hawaii, China and Taiwan. Many A. mangium plantations have been established in acidic soil or abandoned land or Imperata grassland, for example, in Bangladesh (Latif et al., 1995); in Sabah (Latif et al., 1995; Williams et al., 1992) and in Serdong (Majid et al., 1994; Awang, 1994) in Malaysia; Sangmelina, Cameroon, Kenya (Dugurna et al., 1994); Skaerat, Thailand (Khemnark, 1994); Hawaii, USA (Cole et al., 1996); Bogor (Anwar, 1992; Wibowo et al., 1992), Paseh and Kadipaten (Widiarti and Alrasjid, 1987) in Indonesia; Bengal, India (Basu et al., 1987), etc.
Indonesia, with some of the world's largest paper and pulp mills, has been increasingly relying on plantations as the source of wood and A. mangium is the preferred choice. Asia Paper and Pulp group has two affiliate companies with a total concession of 540,000 hectares. By 1996, one company had planted 123,000 hectares of A. mangium, about 90% of all its plantation, which represented 180 million seedlings. It is estimated that by 2004, Asia Paper and Pulp group will virtually source all its wood from plantation, mainly A. mangium plantation (Bayliss, 1998a; Bayliss, 1998b).
Besides its use for paper and pulp, A. mangium timber can be used for other applications such as cement banded particleboard, plywood and decorative panel manufacture (Yusoff et al.,
A large scale of mono-plantation of vegetative propagation from a single plant faces a higher risk of disease infection. It has been found that diseases spread very fast in this kind of plantation and cause large economic losses. Many diseases devastate A. mangium: Cinnamon fungus (Phytophehera cinnamomi) infection results in collapse and death of plants; Seedling blight, defoliation and dieback due to Glomerella cingulata caused serious losses to A. mangium in nurseries. Leaf spot by Cyclindrocladium quinqueseptatuni causes defoliation of seedlings and young trees; Powdery mildew (Oidiunr spp) severely affected A. mangium seedlings in nurseries in Thailand; Red rot diseases, caused by ganoderma sp. affects A. mangium in Malaysia; Brown root disease caused by Phellinus noxium affects A. mangium in Malaysia and the Solomon islands (Simmons, 1987; Gutteridge and Shelton, 1994). Traditional breeding has been less successful in getting disease resistance in A. mangium, mostly likely due to lack of such a gene in the natural gene pool. Molecular breeding by introducing foreign disease resistance genes becomes an important option.
Studies on A. mangium tissue culture have been limited to micropropagation (Bhaskan and Subbash, 1996; Ahmad, 1991; Galiana et al., 1991a; Galiana et al., 1991b). A combination of traditional breeding of elite trees and techniques of large-scale vegetative propagation make the large-scale plantation possible. There has been no report on regeneration or genetic transformation of A. mangium. Our invention describes the conditions for regeneration of whole plant via organogenesis and a genetic transformation system.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
SUMMARY OF THE INVENTION
A. System of regeneration from callus in Acacia mangium 
Different parts of Acacia mangium seedlings or vegetative micropropagated plantlets (hypocotyl, cotyledon, leaflet, petiole and stem) were used as explants. The seedlings were from isolated seed embryo culture and the plantlets were obtained by micropropagation from meristem regions of a 2-year old tree. Callus formation was induced by MS basic medium (Murashige and Skoog, 1962) supplemented with auxins (either 2,4-D from 0.5 mg/L to 5 mg/L or α-naphthaleneacetic acid (NAA) from 0.5 mg/L to 2 mgtL) and cytokinins (either kinetin (KT) 0.5 mg/L to 3.0 mg/L or 6-benzylaminopurine (6-BA or BA) 0.5 mg/L to 3.0 mg/L).
Adventitious bud induction was achieved on medium AM-265 (MS basic medium with thidiazuron (1-phenyl-31,2,3-thiadiazol-5-yl)urea or TDZ) 1.0 mg/L, indole-3-acetic acid (LAA) 0.25 mg/L, casein enzymatic hydrolysate (CH) 100 mg/L, L-ascorbic acid (vitamin C or Vc) 100 mgfL, L-glutamine (Gin) 150 mg/L, L-asparagine monohydrate (Asn) 150 mg/L, L-proline (Pro) 150 mg/L, pH 5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8%, sucrose 30 g/L) under the growth conditions of {fraction (16/8)} hours light/dark (LID) cycle and at 28° C.
Pinnate leaf formation and adventitious bud elongation were obtained on medium AM-337 (MS basic medium with TDZ 0.01 mg/L, CH 100 mg/L, Vc 100 mg/L, Gin 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8%, sucrose 30 g/L), or AMA4 (MS basic medium supplemented with 6-BA 2 mg/L, CH 100 mg/L, Vc 100 mg/L, Gin 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8%, sucrose 30 g/L) supplemented with gibberellic acid (GAD 0.5-2.5 mg/L. The growth conditions are 16/8 hrs (LID) cycle and at 28° C.
Adventitious buds formed roots and complete plantlets were obtained on MS or SIMS basic medium supplemented with NAA 0.5 to 3.0 mg/L combined with KT 0-0.5 mg/L or IBA (indole butyric acid) 0.01-0.5 mg/L and CH 100 mg/L, Vc 100 mg/L, Gln 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.35%, or agar 1.4%, sucrose 20 or 30 g/L under the growth conditions of 16/8 hrs (LID) cycle and at 28° C. Complete plantlets were transplanted into peat soil:white sand (3:1) in a growth chamber at a photoperiod of 16/8 hrs (LID) and 25° C.
B. System of genetic transformation of A. mangium 
Based on the above established regeneration system, Agrobacterium strain LBA4404 (Ooms et al., 1981) harbouring binary vector pB1121 in which a GUS gene is under regulation of an 800 bp cauliflower mosaic virus promoter was used for genetic transformation of A. mangium. The Agrobacteria with the plasmid were co-cultured with the following explants: (1) Young adventitious shoots vegetatively micropropagated from auxiliary buds dissected from a 2-year old tree. The shoots consisted of young stem and pinnate leaves but no phyllodes. Stem pieces of about 2-3 cm in length after separating pinnate leaf and auxiliary buds were used as explants. (2) Petiole pieces of 1-2 cm in length and leaflet pieces of 0.4−0.5×0.3−0.4 (cm) from seedlings derived from embryo culture were used as explants. The explants were pre-cultured on AM-265 for three days at 16/8 hrs (UJD) cycle and at 28° C. before infection with activated Agrobacterium for 15 minutes. Infected explants were then cultured for 3 days in the same media in the dark at 22° C. They were then washed and put into AM-265 to induce callus and adventitious buds. Transgenic plants were selected with G418 in media in four stages: G418 12 mg/L for 25 days, G418 20 mg/L for 60 days, G418 30 mg/L for 25 days and then on G418 12 mg/L. Adventitious bud elongation was induced by AM-265 with GA3 2.5 to 5 mg/L in the presence of G418 12 mg/L. For pinnate leaf formation, adventitious buds were transferred into AM41 with GA3 2.5 mg/L at growth conditions of {fraction (16/8)} hours (L/D) cycle and 28° C. Regenerated shoots were excised and checked for GUS staining and 16% tested positive. Putative transgenic adventitious buds were transferred into AM-357 or AM451 with or without G418 (10 mg/L) at the photoperiod of 1618 hours (LID) to induce rooting. Southern blot hybridization showed positive results when NPTII gene was used as probe.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-E show adventitious bud induction from leaf. FIG. 1A shows callus induction. FIG. 1B shows adventitious bud recovery. FIGS. 1C, 1D and 1E show plumule formation and adventitious bud elongation.
FIGS. 2A-C show Acacia mangium regeneration from petiole. FIG. 2C shows callus induction. FIG. 2B shows adventitious bud recovery. FIG. 2C shows plumule formation and adventitious bud elongation.
FIGS. 3A-E demonstrate Acacia mangium regeneration from stem pieces. FIG. 3A shows regeneration from callus. FIG. 3B shows adventitious buds induced from callus. FIG. 3C show root formation, FIG. 3D shows a complete plantlet and FIG. 3E shows a one month old plantlet in pot soil.
FIG. 4 is a map of pBI121 with an 800 bp HindIII-BamHI fragment containing the cauliflower mosaic virus (CaMV)35S promoter cloned upstream of the GUS gene.
FIGS. 5A-C demonstrate rejuvenation of a tree. FIG. 5A shows a two-year old tree.
FIG. 5B shows adventitious bud induction. FIG. 5C shows propagated adventitious buds with plumules.
FIG. 6A shows adventitious buds and FIG. 6B shows stem pieces as explants for transformation.
FIG. 7 demonstrates selection and induction of putative transgenic adventitious buds.
FIGS. 8A and 8B illustrate GUS staining of adventitious buds after selection for 5 months.
FIGS. 9A-C illustrate GUS staining of young transgenic stem pieces. FIGS. 9A-B show stem pieces and FIG. 9C shows a shoot.
FIGS. 10A-C show GUS staining of transgenic leaf and leaf pieces.
FIG. 11 shows the results of a Southern blot to a NPTII probe. 20 μg of DNA was digested with HindIII, run on a gel and blotted. Lanes 1-6 are transgenic lines. Lane ck is a negative control using DNA from a non-transgenic plant Lane 7 is a positive control using DNA from a tomato transgenic line by plasmid pWS42 with NPTh as the selection marker.



DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to methods for genetically transforming tissue of trees, especially Acacia mangium, and regenerating whole plants via organogenesis from the transformed tissue.
The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.
EXAMPLE 1










Reagents and Culture Media







A. MS basic medium: MS medium (Murashige and Skoog, 1962)












Concen-
Sigma




tration
Cat.


Name
Molecular formula
(mg/L)
No.





Macronutrients


Ammonium Nitrate
NH4NO3
1,650
A-3795


Potassium Nitrate
KNO3
1,900
P-8291


Calcium Chloride dihydrate
CaCl2.2H2O
440
C-2536


Magnesium Sulfate
MgSO4.7H2O
370
M-7774


Heptahydrate


Potassium phosphate
KH2PO4
170
P-8416


monobasic, anhydrous


Ferrous Sulfate Heptahydrate
FeSO4.7H2O
27.8
F-8263


Ethylenediamine-tetraacetic
C10H14N2O8Na2.
37.3
E-6635


acid (EDTA)
2H2O (Na2EDTA)


Micronutrients


Potassium Iodide
KI
0.83
P-8166


Boric Acid
H3BO3
6.2
B-9645


Manganese Sulfate
MnSO4.H2O
16.9
M-7899


monohydrate


Zinc Sulfate
ZnSO4.7H2O
8.6
Z-1001


Molybdic Acid (Sodium
Na2MoO4.2H2O
0.25
M-1651


salt:dihydrate)


Cupric Sulfate
CuSO4.5H2O
0.025
C-3036


(Pentahydrate)


Cobalt Chloride
CoCl2.6H2O
0.025
C-2911


(hexahydrate)


Organic Reagents


myo-Inositol
C6H12O6
100
I-3011


Nicotinic acid
C6H5NO2
0.5
N-0765


Glycine
C2H5NO2
2.0
G-6143


Thiamine (Vitamin B1)
C12H17ClN4OS.HCl
0.1
T-3902


Pyridoxine (Vitamin B6)
C8H11NO3.HCl
0.5
P-9755


Hydrochloride










B. Other Organic Reagents











Sigma Cat.


Name
Molecular formula
No.





L-Ascorbic acid (Vitamin C)
C6H8O6
A-2174


Casein Enzymatic Hydrolysate (CH)

C-7290


L-Glutamine (Gln)
C5H10N2O3
G-9273


L-Asparagine monohydrate (Asn)
C4H8N2O3.H2O
A-4284


L-Proline (Pro)
C5H9NO2
P-4655


Sucrose

S-5390










C. Plant Growth Regulators









Sigma Cat.


Name
No.





Indole-3-acetic Acid (IAA)
I-2886


α-Naphthaleneacetic Acid (NAA)
N-0640


1-Phenyl-3-(1,2,3-thiadiazol-5-yl)Urea (Thidiazuron, TDZ)
P-6186


6-Benzylaminopurine (6-BA)
B-3408


Kinetin (KT)
K-0753


Gibberellic Acid (GA3)
G-7645










D. Agar









Sigma Cat.


Name
No.





Type M
A-4800


Purified
A-7921


Phytagel
P-8169










E. Antibiotics.








Timentin (T)
Beecham Pharmaceuticals (Pte) Ltd


Kanamycin (K)
Sigma


Genetins (G418 Sulfate)
Clontech 8056-2










F. Media for callus inductlon










AM-5
MS + 2,4-D 2.0 mg/L + KT 3.0 mg/L



AM-6
MS + 2,4-D 2.0 mg/L + KT 0.5 mg/L



AM-7
MS + NAA 2.0 mg/L + KT 3.0 mg/L



AM-8
MS + NAA 2.0 mg/L + KT 0.5 mg/L



AM-14
MS + 2,4-D 0.5 mg/L + 6-BA 3.0 mg/L



AM-15
MS + 2,4-D 1.0 mg/L + 6-BA 3.0 mg/L



AM-16
MS + 2,4-D 2.0 mg/L + 6-BA 3.0 mg/L



AM-17
MS + 2,4-D 0.5 mg/L + 6-BA 0.5 mg/L



AM-18
MS + 2,4-D 1.0 mg/L + 6-BA 0.5 mg/L



AM-19
MS + 2,4-D 2.0 mg/L + 6-BA 0.5 mg/L



AM-20
MS + 2,4-D 0.5 mg/L + 6-BA 1.0 mg/L



AM-21
MS + 2,4-D 1.0 mg/L + 6-BA 1.0 mg/L



AM-22
MS + 2,4-D 2.0 mg/L + 6-BA 1.0 mg/L



AM-27
MS + NAA 0.5 mg/L + 6-BA 3.0 mg/L



AM-28
MS + NAA 1.0 mg/L + 6-BA 3.0 mg/L



AM-29
MS + NAA 2.0 mg/L + 6-BA 3.0 mg/L



AM-30
MS + NAA 0.5 mg/L + 6-BA 0.5 mg/L



AM-31
MS + NAA 1.0 mg/L + 6-BA 0.5 mg/L



AM-32
MS + NAA 2.0 mg/L + 6-BA 0.5 mg/L



AM-33
MS + NAA 0.5 mg/L + 6-BA 1.0 mg/L



AM-34
MS + NAA 1.0 mg/L + 6-BA 1.0 mg/L



AM-35
MS + NAA 2.0 mg/L + 6-BA 1.0 mg/L



AM-231
MS + 2,4-D 5.0 mg/L + KT 0.5 mg/L



AM-233
MS + 2,4-D 0.5 mg/L + KT 1.0 mg/L



AM-234
MS + 2,4-D 1.0 mg/L + KT 1.0 mg/L










G. Media for adventitious buds induction

    a., AM-265: MS basic medium with TDZ 1.0 mg/L, IAA 0.25 mg/L, CH 100 mgJL, Vc 100 mg/L, Gln 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8% (A-4800, Sigma), sucrose 30 g/L.
    b. AM-261: MS basic medium with TDZ 1.0 mg/L, IAA 0.5 mg/L, CH 100 mg/L, Vc 100 mg/L, Gln 150 mg/L, Asn 150 mgJL, Pro 150 mg/L, pH 5.8 after autoclave, phytagel 0.275 or 0.30%, or agar 0.8% (A-4800, Sigma), sucrose 30 g/L.
    c. AM-304: MS basic medium with TDZ 2.0 mg/L, IAA 0.25 mg/L, CH 100 mg/L, Vc 100 mg/L, Gln 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclave, phytagel 0.275 or 0.30%, or agar 0.8% (A4800, Sigma), sucrose 30 g/L.
    d. AM-30B: MS basic medium with TDZ 1.0 mg/L, LAA 2.0 mg/L, CH 100 mg/L, Vc 100 mg/L, Gln 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8% (A-4800, Sigma), sucrose 30 g/L.

H. Media for pinnate leaf formation

    a. AM-337-MS basic medium with TDZ 0.01 mg/L, CH 100 mg/L, Vc 100 mg/L. Gin 150 mg/L, Asn 150 mg/l and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8% (A4800, Sigma), sucrose 30 g/L.
    b. AMA41: MS basic medium supplemented with 6-BA 2 mg/L, CH 100 mg/L, Vc 100 mg/L, Gin 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.275 or 0.30%, or agar 0.8%, sucrose 30 g/L.

I. Media for root formation

    a. Modified AM-8: MS basic medium with NAA 2.0 mg/L, KT 0.5 mg/L, CH 100 mg/L, Vc 100 mg/L, Gin 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.30%, sucrose 30 g/L.
    b. AM-357: 1⁄2MS basic medium with NAA 2.0 mg/L, KT 0.5 mg/L, CH 100 mg/L, Vc 100 mg/L, Gin 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.30%, sucrose 30 g/L.
    c. AM451: 1⁄2MS basic medium with NAA 2.0 mg/L, KT 0.1 mg/L, CH 100 mg/L, Vc 100 mg/L, Gin 150 mg/L, Asn 150 mg/L and Pro 150 mg/L, pH 5.8 after autoclaving, phytagel 0.35%, sucrose 20 g/L.

J. Transplantation
    
        Peat soil:white sand 3:1
    
    












K. Media for Agrobacterium tumefaciens activation

















LB medium




Tryptone
1.0%


Yeast extract
0.5%


NaCl
0.8%







pH 7.0 before autoclaving (Sambrook et al., 1989)


YEP medium (Per liter) (Chilton et al., 1974)









Bactopeptone
10
g


Yeast extract
10
g


NaCl
5
g


AB medium


20 × Phosphate Buffer (per liter)


K2HPO4
60
g


NaH2PO4
20
g


Autoclave this solution separately


20 × Salts solution (per liter)


NH4Cl
20
g


MgSO4.7H2O
6
g


KCl
3
g


CaCl2
0.2
g


FeSO4.7H2O
0.05
g


pH to 7.0 before autoclaving







To make up the final medium, combine (to a final volume of 1 liter):









50 mL 20 × Phosphate Buffer




50 mL 20 × Salts Solution


900 mL sterile ddH2O


Induction medium


MES buffer, pH 6.0
 30
mM


1 × AB medium


Glucose
0.5%


Acetosyringone
100
μM (a stock solution




of acetosyringone




must be made up




fresh in DMSO)









EXAMPLE 2
Assay for GUS Activity
Stain the tissue overnight at 37° C. in GUS staining solution. Gus staining solution is described by Jefferson (1987). It is X-gluc-1 mM, sodium phosphate (pH 7.0)-100 mM, EDTA-10 mM, and Triton X-100-0.1% GUS staining showed a positive blue reaction in adventitious buds and stem and leaf (FIGS. 8, 9, and 10).
EXAMPLE 3
Southern Analysis
A method for performing Southern blots is described in Sambrook et al. (1989). The method is:
A. DNA extraction
2-5 g fresh sample was frozen in liquid N2. This was ground with a mortar and pestle in liquid nitrogen to a fine powder. The powder was transferred to a centrifuge tube (50 mL). 15 mL of Extraction Buffer was added, 2 mL 10% SDS was added and mixed thoroughly. This was incubated at 65° C. for 15 minutes. 5 mL S M KAc was added and shaken vigorously. The mixture was incubated in ice for 20 minutes and then spun at 25,000×g for 20 minutes. The supernatant was filtered through Microcloth into a new tube. The DNA was precipitated with 1⁄2 volume of isopropanol, mixed and incubated at −20° C. for 30 minutes.
The DNA was pelleted at 25,000×g for 30 minutes, the supernatant was poured off, and the tube was inverted and air dried for 30 minutes. The pellet was dissolved with 0.7 mL of 1×TE (pH 8.0) and transferred to an Eppendorf tube. This was spun 10 minutes. The supernatant was transferred to a new tube, 7 μL RNase (10 mg/mL) was added and left at room temperature for 10 minutes, then 75 μL 3 M NaAc (pH 5.3) and 500 μL of isopropanol were added. The solution was mixed and the DNA was pelleted in a microcentrifuge at full speed for 5 minutes. The pellet was washed with 70% ethanol, air dried and dissolved with 100 μL 1×TE (pH 8.0).










B. Extraction buffer



















100 mM Tris-HCl, pH 8
50 mL




50 mM EDTA, pH 8
50 mL



500 mM NaCl
50 mL



10 mM β-ME
0.6 mL



ddH2O
to 500 mL










C. Enzyme digestion

Reaction system 1: DNA sample 100 μL (20 μg), 10×HindIII buffer 40 μL, HindIII 8 μL (80 units), add sterile double distilled H2O 252 μL to total volume 400 μL.
The reaction was incubated at 37° C. for overnight. 40 μL of 3 M NaAc (pH 5.3) and 2⁄3volume of 100% ethanol were added to the reaction system and this was incubated at −20° C. for 30 minutes. This was spun at full speed at 4° C. for 20 minutes. The supernatant was poured off and the tube was air dried for 30 minutes then the pellet was dissolved in 30 pL sterile double distilled water.
D. Electrophoresis
Electrophoresis was performed on a 0.8% agarose gel in 1×TBE at 28V for overnight.
E. Transfer of DNA from Agarose gels to Nylon Membranes
This step was performed as described in Sambrook et al. (1989).
F. Prehybridization
A prehybridization solution of 6×SSC, 5× Denhardt's reagent, 0.5% SDS, 100 μg/mL denatured, fragmented salmon sperm DNA (Stratagene products) and 50% formamide was prepared. 50× Denhardt's is: 5 g of Ficoll, 5 g of polyvinylpyrrolidone, 5 g of bovine serum albumin and ddH2O to 500 mL, filtered and stored at −20° C.
After fixing DNA to the membrane, the membrane was placed into a hybridization tube containing suitable prehybridization solution using 0.2 mL prehybridization solution for each square centimeter of nylon membrane. The membrane was incubated at 42° C. for 6 hours.
G. Probe label
During the prehybridization, labeled probe was prepared using a Boehringer Mannheim-High Primer DNA Labeling Kit. 50 ng of NPTII were added then brought to a volume of 8 AL with H2O. The DNA was denatured in a 100° C. heat block for 10 minutes, chilled quickly in ice, and pulse spun.
On ice the denatured DNA was mixed with: 4 AL High Prime reaction mixture, 3 PL of dATP, dGTP, dTTP mixture, and 5 gL of α-32P dCTP, 3000 Ci/mmol (Biolab). This was incubated at 37° C. for 10 minutes. The reaction was stopped by adding 20 μL of 50 mM EDTA (pH 8.0).
The labeled probe was purified by running through a small Sephadex G50 column prepared on a small Pasteur pipette. The eluent was monitored with a counter and the first peak was collected.
H. Hybridization
The probe was added into the hybridization tube, then incubated at 42° C. for 10-24 hours.
I. Washing membrane
The hybridization solution was poured off and the membrane was submerged in 2×SSC, 0.5% SDS at room temperature for 10 minutes. The membrane was transferred into 2×SSC, 0.1% SDS at room temperature for 15 minutes. The solution was replaced with 0.5×SSC, 0.1% SDS and the membrane was incubated at room temperature for 15 minutes. The solution was replaced with 0.1×SSC, 0.1% SDS and incubation continued at 55° C. for 30-55 minutes. The membrane was transferred into 0.1×SSC at room temperature for 3-5 minutes, then air dried on 3MM Whatman paper for 30 minutes.
J. Autoradiography
The membrane was exposed to X-ray film (Kodak,) to obtain an autoradiographic image at −80° C. for one day or more.
Using NPTII fragment as a probe, Southern blotting showed that the NPTII gene had integrated in adventitious buds (FIG. 11). The results demonstrate that this protocol of Acacia mangium transformation is very successful.
EXAMPLE 4
Regeneration of A. mangium 
A. Embryo culture
Mature seeds (black coat) were pre-treated with 98% H2SO4 for 2-3 minutes and washed with tap water several times. Treated seeds were sterilized with 70% ethanol for 2-3 minutes and vashed 5 times with sterile ddH2O. Seeds were then immersed in 0.1% HgCl2 for 6 minutes and washed 5 times with sterile ddH2O, again sterilized with bleach 30% (market product) for 6 minutes, then washed 5 times with sterile ddH2O. Sterilized seeds were soaked in sterile ddH2O overnight for isolating zygotes for embryo culture. MS basic medium (Murashige and Skoog, 1962), pH 5.8, sucrose 30 g/L, phytagel 0.25% or agar 0.7% (Sigma, A4800), with or without activated charcoal, was used to culture mature embryo. Isolated mature zygotic embryos were cultured on MS using a photoperiod of {fraction (12/12)} hours or {fraction (16/8)} hours (LID) or complete dark, at 25-28° C. Germinated hypocotyls or leaves or petioles or stems were used as explants to induce callus formation.
B. Callusinduction
Different media were used to induce callus, including medium Nos. AM-5, 6, 7, 8, 14, 15, 16, 17, 18, 19, 20, 21, 22, 27, 28, 29, 30, 31, 32, 33, 34, 35, 231, 233 and 234 at {fraction (12/12)} hours or {fraction (16/8)} hours (LID) or Dark at 25° C. Callus induced from young leaflet: AM-L; from young petiole: AM-P; from hypocotyl: AM-H; from young stem: AM-S; from bud: AM-B; from root: AM-R. All above calli were used to induce adventitious buds, using a photoperiod of {fraction (16/8)} hours, 1800-2000 lux, and 28° C. The results of callus induction on different media were different (Table.1). Generally callus induction was not difficult. All explants produced calli at the photoperiod of 12/12 hrs or 16/8 hrs (LID) (see, e.g., FIG. 1A; FIG. 2A; FIG. 3A), and 25° C. for 20 days.







TABLE 1







Results of callus induction on different media


at 12/12 hours (L/D), 25° C. cultured for 35 days













No. of explants



Number of
No. of explants
complete callus


Medium No.
Explants (leaflet)
forming callus
formation













AM-5
40
40
33


AM-6
40
40
31


AM-7
40
40
3


AM-8
40
40
4


AM-14
40
40
36


AM-15
40
40
40


AM-16
40
40
38


AM-17
40
40
36


AM-18
40
40
39


AM-19
40
40
39


AM-20
40
40
38


AM-21
40
40
40


AM-22
40
40
40


AM-27
40
40
12


AM-28
40
40
8


AM-29
40
40
11


AM-30
40
40
10


AM-31
40
40
6


AM-32
40
40
2


AM-33
40
40
4


AM-34
40
40
4


AM-35
40
40
6


AM-231
40
40
30


AM-233
40
40
20


AM-234
40
40
20



Petiole


AM-6
20
20
15


AM-17
20
20
20









C. Adventitious bud induction

Calli induced from leaflet, petiole, young stem and buds were cultured on AM-261, AM-265, AM-304, and AM-308, at a photoperiod of 1618 hrs (LJD), at 28° C. One month later, there were some bud recoveries from callus (FIG. 1B; FIG. 2B). Bud recovery from friable callus first appeared as compact and smooth bud terminal. The ratio of bud recovery reaches 15% on AM-265. But on these media, induced buds could not easily form pinnate leaves and elongate.
D. Pinnate leaf formation and adventitious bud elongation
Bud recoveries from callus were transferred into AM-337 or AM41 with GA, 2.5 mg/L, at a photoperiod of 16/8 hrs (L/D), 1800-2000 lux, and 28°C. After culture for one month, some of them could form pinnate leaves and elongate (FIGS. 1C, D, E; FIG. 2C; and FIG. 3B). The ratio of pinnate leaf formation and bud elongation reaches 16.67%.
E. Root formation
Elongated buds were cultured in AM-357 or AM451 for root formation. 20 days later, adventitious roots began to appear from basic stem of buds (FIG. 3C). After root formation, plantlets were transferred to MS basic medium without any plant growth regulators. Plantlets grow normally and fast, and the root system grew well (FIG. 3D).
F. Transplantation
Phytagel on plantlets was washed away and the plantlets were transferred to pot (peat soil:white sand 3:1) and grown in a growth chamber at a photoperiod of {fraction (16/8)} hours and 25° C. One month later the plantlets were transferred to a green house (FIG. 3E).
EXAMPLE 5
Rejuvenation of Tree
The auxiliary buds of a two-year old young tree (FIG. 5A) were cultured on MS basic medium with NAA 0.1 mg/L, 6-BA 3.0 mg/L, CH 100 mg/L, Vc 100 mg/L, Pro 150 mg/L, Asn 150 mg/L and Gin 150 mg/L, phytagel 0.275% or agar 0.8% (A4800, Sigma), pH 5.8 after autoclaving at 121° C., sucrose 30 g/L. Sixty days later, some adventitious buds with phyllodes were obtained (FIG. 5B). Induced adventitious buds with phyllodes were subcultured on AM-41 (MS basic medium with 0.6-BA 2 mg/L, CH 100 mg/L, Vc 100 mg/L, Pro 150 mg/L, Asn 150 mg/L and Gln 150 mg/L, phytagel 0.275% or agar 0.8% (A4800, Sigrna), pH 5.8 after autoclaving at 121° C., sucrose 30 g/L). After sub-culturing adventitious buds with phyllodes twice in about two months, the rejuvenation of adventitious shoots with pinnate leaf was obtained (FIG. 5C). Adventitious buds can be used as explants for transformation (FIG. 6A).
EXAMPLE 6
A. mangium Genetic Transformation
A. Activation of Agrobacterium tumefaciens Strain LBA44041 pBI121 Agrobacterium strain LBA4404 (Ooms et al., 1981) harbouring binary vector plasmid pB 121 (size of vector 13.0 kb, Clontech, FIG. 4) was used for this experiment. pB 121 was derived from pBl 101 with an 800-bp HindIII-BamnHI fragment containing the cauliflower mosaic virus (CaMV) 35S promoter cloned upstream of the GUS gene. From a permanent glycerol stock stored at −70° C., Agrobacterium lumefaciens pBI121/LBA4404 was streaked onto a solid LB (pH 7.0) medium plate containing Streptomycin 100 mg/L (Str100) and Kanamycin 50 mg/L (Ks). This was incubated 2-3 days at 28° C. Fresh pBI121/LBA4404 was streaked onto solid LB medium (pH 7.0) with Streptomycin 100 mg/L (Str100) and Kanamycin 50 mg/L (K50) for overnight or 24 hours at 28° C. in the dark. Several colonies of pBI121/LBA4404 were picked and pBI121/LBA4404 was inoculated in liquid 50 mL LB medium with Streptomycin 100 mg/L (Str100) and Kanamycin 50 mg/L (K50), and cultured at 28° C. under dark, 250 rpm for 10 hours until OD600=0.70−1.10. This was spun down at 3500 rpm for 30 minutes or 5000 rpm for 10 minutes, and resuspended in 4-5 volumes of YEP medium (pH 7.0) with Streptomycin 100 mg/L (Str100) and Kanamycin 50 mg/L (WO), OD600=0.10-0.20, incubated at 28° C. in the dark, 250 rpm for 8-10 hours, OD600=0.70-1.20. This was spun down at 3500 rpm for 30 minutes and resuspended in the same volume of sterile NaCl 0.9%. This was spun down at 3500 rpm for 30 minutes and resuspended in 2-3.5 volumes of induction medium, OD600=0.2-0.3, and incubated at 28° C. in the dark, 120 rpm for 8-15 hours, OD600=0.70-1.20 for infecting explants or callus or a cell suspension.
B. Preculture of stem pieces
Stem pieces were cultured on AM-265 for 3 days using a photoperiod of 16/8 hrs (L/D), 1800-2000 lux, at 28° C.
C. Co-cultivation
After preculture, stem pieces were soaked in 0.5 M mannitol for 20-25 minutes and then transferred to activated pBI121 ILBA4404 suspension for 15 minutes. Infected young stem pieces were washed once and dried on sterile Whatman paper before being cultured on AM-265 with 100 μM acetosyringone at pH 5.2 at 22° C. in the dark for 3 days.

    D. Selection of transformed adventitious buds and transformed callus


After co-culturing for 3 days, stem pieces were washed with sterile ddH2O for 10 times and dried on sterile Whatman paper. Then stem pieces were cultured on AM-265 containing Timentin 250 mg/L and 0418 12 mg/L, phytagel 0.275%, using a photoperiod of 16/8 hrs (LID) and 28° C. for selecting transformed callus or transformed adventitious buds. Subsequently, stem pieces were cultured on the above media with G418 12 mg/L for 25 days, G418 20 mg/L for 60 days, G418 30 mg/L for 25 days, then on G418 12 mg/L. After continuous selecting for 4 months on antibiotics, 33.75% of stem pieces formed adventitious buds, and 2.5 mg/L to 5 mg/L GA3 was added into the same media to promote adventitious bud elongation (FIG. 7). After selection for five months, Timentin was no longer used in the medium and some adventitious buds were used for GUS staining. GUS staining showed positive blue colour reaction in adventitious buds (FIG. 8, FIG. 9, and FIG. 10). Adventitious buds were transferred to AM-41 with GA3 2.5 mg/L to promote pinnate leaf formation.
E. Root formation
Transformed adventitious buds were transferred into AM-357 or AM451 with or without G418 (10 mg/L) using a photoperiod of 16/8 hrs (L/D) at 28° C.
While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.
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## Claims

1. A method of transforming Acacia mangium with a gene of interest comprising the steps of:
a) activating Agrobacterium tumefaciens comprising said gene of interest by culturing said Agrobacterium in induction medium comprising acetosyringone; 
b) preculturing an explant of Acacia mangium selected from the group comprising a stem, a leaflet, a petiole and a bud in medium comprising supplemented basic MS medium, wherein said explant is soaked in 0.5 M mannitol prior to the co-cultivation; 
c) co-cultivating said activated Agrobacterium tumefaciens and said precultured explant in medium comprising supplemented basic MS medium to produce infected explants; 
d) culturing said infected explants in medium comprising supplemented basic MS to induce callus and adventitious buds; and 
e) culturing said callus or adventitious buds on a selective medium comprising supplemented basic MS medium; 
wherein in said supplemented basic MS medium comprises a) thidiazuron, b) indole-3-acetic acid, c) casein enzymatic hydrolysate, d) L-ascorbic acid, e) L-glutamirie, f) L-asparacine, g) L-proline, h) sucrose and l) phytagel or agar.

2. The method of claim 1 wherein said co-cultivating is performed in the dark.

3. The method of claim 1 wherein said preculture is performed using a photoperiod of 16 hours light/8 hours dark.

4. The method of claim 1 wherein said culturing on selective medium is performed using a photoperiod of 16 hours light/8 hours dark.

5. A method for promoting elongation of transformed adventitious buds of Acacia mangium comprising transforming an Acacia mangium explant by the method of claim 1 and further comprising a step of addition of gibberellic acid to the culture medium following formation of adventitious buds.

6. A method for promoting pinnate leaf formation on transformed adventitious buds of Acacia mangium comprising transforming an Acacia mangium explant by the method of claim 1 and further comprising culturing adventitious buds which develop on a medium with gibberellic acid.

7. A method of preparing transgenic Acacia mangium cells comprising the steps of
a) preculturing stem pieces of Acacia mangium in culture medium comprising supplemented basic MS medium, wherein said stem pieces are soaked in 0.5M mannitol prior to co-cultivating with Agrobacterium tumefaciens wherein said supplemented basic MS medium comprises a) thidiazuron, b) indole-3-acetic acid, c) casein enzymatic hydrolysate, d) L-ascorbic acid, e) L-glutamine, f) L-asiparagine, g) L-proline, h) sucrose and i) phytagel or agar; and 
b) co-cultivating said stem pieces of step (a) with Agrobacterium tumefaciens in culture medium AM-265, wherein said Agrobacterium was activated by culturing the Agrobacterium in induction medium comprising acetosyringone prior to said co-cultivation.

8. The method of claim 7 wherein said preculturing is performed for 3 days using a photoperiod of 16/8 hours (light/dark).

9. The method of claim 8 wherein said preculturing is performed using 1800-2000 lux for the light cycles.

10. The method of claim 7 wherein said preculturing is performed at 28° C.

11. The method of claim 7 wherein said Agrobacterium tumefaciens was activated by growing the Agrobacterium in induction medium, at 28° C. in the dark.