A plant transformation system designed for high throughput genomics in Gossypium hirsutum to study root–organism interactions

Figures & data

Table 1. PCR primers. The primers used are A. rhizogenes VirG (Ar-VirG), G. hirsutum S21 (Gh-S21), G. max NPR1-2 (Gm-NPR1-2), and eGFP.

Primer name	Primer application	Product size	Primer (5′→3′)
eGFP	PCR	717	F: GAATTTGTTTCGTGAACTATTAGTTGCGG
	PCR		R: GCATGCCTGCAGGTCACTGGATTTTG
eGFP	qPCR	134	F: AGGAGCTGTTCACCGGGG
	qPCR		R: GGTGCAGATGAACTTCAGGGTC
	qPCR probe		P: AGTTCAGCGTGTCCGGCGAGG
Ar-VirG	PCR	690	F: ATGCGCCATCTTATTACCGAGTATTTAAC
	PCR		R: TCAGGCCGCCATCAGACC
Gm-NPR1-2	PCR	1773	F: CACCATGGCTTATTCAGCCGAACCC
	PCR		R-TTACACTTTCCTAGCCTTGTAATGTACA
Gm-NPR1-2	qPCR	104	F: TGATGCTGACCTTGTTGTCG
	qPCR		R: ATGACCCCTTCTCCCTCTTG
	qPCR probe		P: CATCGATGTATTCTGGCCTCTAGGAGTAAG
Gh-S21	qPCR	146	F: ATGAACGCTATATCGAGGCGA
	qPCR		R: AACGCTTGCTCCAAGTTGC
	qPCR probe		P: CAGCGAGACAGGGGATCAATTTATTGA

Figure 1. pRAP15 vector. Legend: open reading frame (ORF), pink; ORF1, homologous to partitioning protein A (ParA); ORF2, no significantly homologous leader sequence 5′ fused to the Reporter gene (enhanced green fluorescent protein [eGFP]), green. ORF3, homologous to resolvase; ORF4, homologous to replicase A (RepA); ORF5, homologous to tetracycline resistance gene (TetR); ORF6, homologous to aminoglycoside adenyltransferase. Origin of replication (pBR322), black. Other gene (ccdB, attR1, attR2), magenta. Selectable marker (chloramphenicol [CAT]), orange. Terminator (cauliflower mosaic virus [CaMV] 35S), brown. Blue lettering, unique restriction sites.

Figure 2. Confirmation of A. rhizogenes being genetically transformed with pRAP15. Lane 1, DNA ladder. Lane 2, eGFP; Lane 3, Ri VirG; Lane 4, Gm-NPR1-2. Numbers in lane 1 represent the number of base pairs.

Figure 3. Cocultivation. (A) The roots of G. hirsutum being removed in a Petri dish containing genetically transformed A. rhizogenes. (B) The root-less G. hirsutum are placed into a beaker containing genetically transformed A. rhizogenes. (C) Root-less G. hirsutum in beakers prior to vacuum infiltration. (D) Root-less G. hirsutum in vacuum infiltration chamber prior to infiltration. (E) G. hirsutum planted in vermiculite after vacuum infiltration. (F) Twelve humidity chambers under lights, each containing 50 G. hirsutum plants that are recovering from the transformation procedure.

Figure 4. eGFP expression in transgenic G. hirsutum roots. Lane 1, DNA ladder; Lane 2, eGFP.

Figure 5. qPCR to determine the expression of the transgene in RNA extracted from transgenic roots. Left panel, roots transformed with the empty pRAP15 vector. Right panel, roots transformed with Gm-NPR1-2.

Table 2. A time line for production of composite G. max and G. hirsutum plants with transformed roots.

Step number	Step	G. max	G. hirsutum
1	Seed germination efficiency	98%	98%
2	Age of seedling used for plant transformation procedure	7 days	14 days
3	Survival rate-post transformation	95%	98%
5	Days plantlets are grown in culture room prior to transfer to greenhouse	14 days	28 days
6	Days plantlets are grown in greenhouse prior to removing nonengineered roots	14 days	30 days
7	Transformation efficiency	82%	91%
8	Days plantlets are grown in greenhouse after removal of nonengineered roots	14 days	14 days
Total days		49	86

Figure 6. Target region of Gm-NPR1 for qPCR study. The G. raimondii NPR1 homologs are Gorai.011G050200.1 and Gorai.006G091900.1 as compared to G. max Glyma09g02430.1. Cyan, forward primer; red, qPCR probe; magenta, reverse primer. Asterisks represent identical nucleotides presented only in the primer regions.

Figure 7. G. hirsutum grown in 6 inch pots that are of sufficient size for experiments examining root biology. Left, a control, G. hirsutum pRAP15-engineered plant lacking an engineered transgene (empty vector). Right, G. hirsutum engineered with a Gm-NPR1-2 transgene.

Figure 8. G. hirsutum eight weeks after transformation procedure. (A) Control G. hirsutum that is altogether untransformed and lacking fluorescence. (B) G. hirsutum that is transformed with Gm-NPR1-2 as indicated by the fluorescence of the visible reporter eGFP. Bars = 1 cm.