Chapter 4, Transformation of broccoli and Arabidopsis to alter ethylene and cytokinin biosynthesis 1 07
was then digested with Eco RI and the earlier prepared AS promoter was
cloned into these blunt-ended sites. This vector was named pAS/BoAC02, and was sequenced in both directions using the T3 and T7 primer sites of the vector, to check orientation of the two inserted fragm ents.
Ill. The AS-BoAC02 insert was removed from pASIBoAC02 by Eco RI-Xba I digestion, and cloned into the Eco RI-Xba I sites of pART7 (Gleave, 1 992). This vector was named pART7/AS-BoAC02.
IV. Eco RI-Not I digestion ofpART7/AS-BoAC02 yielded the 3.9 kb fragment containing the chimeric gene construct AS-BoAC02-0CS3'. This insert was then blunt-ended and cloned into the blunt-ended Not I site of pART27 (Gleave, 1 992) to form the new binary vector pPN 1 O.
4.2.2.2 pPN 1 1 a n d pPN 1 09 - SAG12-IPT-NOS3' The pUC 1 8 based vector pSGS 1 6 was donated by Dr Richard Amasino, and contained the chimeric gene SAG1 2-IPT-NOS. The chimeric gene was removed from pSGS 1 6 by digesting with Sph I -Sma 1 . The
released 3.2 kb fragment was then cloned into the Sph I and Eco RV sites of pGEMSzf
vector to produce another Not I site at the terminator end of the chimeric gene. This
vector was named pGEMS/SAGI 2-IPT. Not I digestion of this vector yielded the 3.2 kb
chimeric gene, which was cloned into the Not I sites of pBJ49 to produce the binary
vector pPN 1 1 and into pART27 to produce the binary vector pPN 1 09.
4.4.2.3 pPN1 and pPN1 1 0 - MYB3os-IPT-TERM The pBLUESCRIPT based vector pGAK4
contained the chimeric gene MYB30S-IPT-TERM. Not I digestion of this vector yielded the 6kb fragment containing the chimeric gene. This Not I fragment was cloned into Not I digested pBJ49, to produce the binary vector pPN 1 and into pART27 to produce the binary vector pPN 1 1 O.
4.2.2.4 pPN1 1 1 - MYB3os-IPT-TERM : :AS-BoAC02-0CS3' pART7/AS-BoAC02 was digested with Eco RI and this site was then blunt-ended using Bacteriophage T4 DNA
polymerase, and Not I adapters (New England Biolabs) were ligated to this blunt site.
This linearised vector was then digested with Not I to yield the 3 . 9 kb AS-BoAC02-
Chapter 4, Transformation of broccoli and Arabidopsis to alter ethylene and cytokinin biosynthesis 1 08
was named pGEM/AS-BoAC02-0CS3', and was digested with Not I to yield the 3.9 kb
AS-BoAC02-0CS3' fragment. pGAK4 was also digested with Not I to yield the 6kb
MYB30S-IPT-TERM fragment. Both these Not I fragments were then cloned into the Not
I site of pART27 in a three-way ligation to produce the binary vector pPN l l 1 .
4.2.2.5 pPN1 1 2 - SAG12-IPT-NOS3': :AS-AC02-0CS3' pART7/AS-BoAC02 was digested with Eco RI and this site was then blunt-ended using Bacteriophage T4 DNA
polymerase. The SAGI 2-IPT-NOS3' fragment of pSG5 1 6 was liberated by digestion with Sma I-Sph I. The Sph I site was blunt-ended using the Klenow fragment of E. coli DNA polymerase I. The blunt 3.2 kb SAGI 2-IPT-NOS3' fragment was then cloned into the blunt-ended Eco RI site of pART7/AS-BoAC02. Not I digestion liberated a 7. 1 kb
SAG12-IPT-NOS3'::AS-BoAC02-0CS3' fragment which was cloned into the Not I site
of pART27 to produce the binary vector pPN1 1 2 (Gleave, 1 992).
4.2.3 Transformation
4.2.3.1 Broccoli Media used :
• Seed germination medium - 1 x MS salts, B5 vitamins, 3% sucrose (v/v), 0.8 % Phytagar (w/v), pH 5.7, poured into medium tubs ( 50 mm deep, 90 mm diameter).
• Co-cultivation medium - 1 x MS salts, B 5 vitamins, 3% sucrose (v/v), 1 3.3 IJM 6-benzyl
amino purine (6-BAP) (3 mg
L-1),
0.8 % Phytagar (w/v), p H 5.7, poured into petri d ishes (20 mm deep, 90 mm diameter).• Selection medium - 1 x MS salts, B5 vitam ins, 3% sucrose (v/v), 3 mg
L-1
6-benzyl-aminopurine (6-BAP), 0.7 mM ticarciliin disodium (Timentin) (300 mg
L-\
0.8 % Phytagar (w/v), pH 5 . 7 , containing either 42.8 IJM kanamycin (25 mg L-\ or either 3 . 8 or 1 4.3 IJMhygromycin B (2 or 7.5 mg
L-\
poured into plates (20 mm deep, 90 mm round).• Rooting medium - 1 x MS salts, B5 vitamins, 3% sucrose (v/v), 0.35 m M ticarcillin disodium
(Timentin) ( 1 50 mg L-1), 0.8 % Phytagar (w/v), pH 5.7, containing either 85.6 IJM kanamycin (50 mg
L-\
or 3.8 IJM hygromycin B (2 mgL-\
poured into medium tubs.• Bacterial m in imal culture medium - 7.6 m M (N H4hS04, 1 .7 m M sodium citrate, 78.7 mM
K2HP04, 0.33 M KH2P04, 1 m M MgS04, 0.2% sucrose (w/v), p H 7 . 2 .
A cotyledonary petiole and hypocotyl based system was modified from Moloney et al. ( 1 989) and ludith Irwin (personal communication), and used as described by Gapper et al. (2002), and i s shown by flow diagram in Figure 4.3. Seeds were surface sterilised in
1 09
Hypocotyl and cotyledonary petiole explants excised from five day old seedlings and inoculated in A. tumefa ciens cu lture (A660 0 . 1 ), were co-cultivated for three days then transferred to selection media containing antibiotics.
Putative transgenic shoots regenerated after between three and eight weeks from inoculated explants, and were transferred to hormone-free rooting media. Plant numbers were amplified clonally by adventious shoot regeneration. Once roots were established plantlets were
exflasked and transferred to soil and grown to maturity in a containment glasshouse.