# US 10,370,671 B2 · Binary vectors with minimized biosafety concerns and high transformation rates by engineered plant-derived transfer-DNA

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- Number: US 10,370,671 B2
- Document ID: US10370671B2
- Status: Granted
- Kind: B2
- Application number: 15/299,380
- Filed: 2016-10-20
- Published: 2017-09-14
- Granted: 2019-08-06
- Earliest claimed: 2016-10-20
- Applicants: Bahar Zare; Mohammad Ali Malboobi; Mohammad Sadegh Sabet Jahromi; Peyman Norouzi
- Inventors: Bahar Zare; Mohammad Ali Malboobi; Mohammad Sadegh Sabet Jahromi; Peyman Norouzi
- Web page: https://patentgrep.com/patent/US10370671B2

## CPC

- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/82: for plant cells {, e.g. plant artificial chromosomes (PACs)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

An engineered plant-derived transfer (P-)DNA was designed and constructed based on a couple of T-DNA homologous sequences in sugar beet genome. Plant transformation efficacy of the engineered P-DNA was analyzed compared to conventional T-DNA in two independent systems, stable transformation of tobacco plant and sugar beet hairy roots. The outcomes demonstrated that plant transformation is directed well by vectors carrying the engineered P-DNA with higher efficiency than the conventional binary vector in both experimental systems. This vector was further improved by adding two matrix attachment regions within P-DNA. This new vector was even more efficient in gene transfer, higher than conventional binary vector.

## Claims

### Claim 1

A binary vector comprising two plant-derived transfer DNA (“P-DNA”), wherein the binary vector comprises:
SEQ ID NO: 1;
a set of ordered unique restriction endonuclease cleavage sites for insertion of the heterologous DNA; and
matrix attachment region sequences between the LB and RB sequences configured to improve transformation rate and gene expression level in plant.

### Claim 2 (depends on 1)

The binary vector of claim 1, wherein the SEQ ID NO: 1 comprises TCTGTTCGGG SEQ ID NO: 4, an optimized engineered DR element.

### Claim 3 (depends on 1)

The binary vector of claim 1, wherein the SEQ ID NO: 1 comprises ATTGATTTTTGATTC SEQ ID NO: 5, an optimized engineered UL element.

### Claim 4 (depends on 1)

The binary vector of claim 1, wherein the LB and RB sequences are selected from sugar beet genome and optimized for higher transformation rate.

### Claim 5

A binary vector comprising a sequence of a plant-derived transfer DNA (“P-DNA”) comprising SEQ ID NO: 1, wherein:
the vector further comprises matrix attachment region sequences between the LB and RB sequences that improve transformation rate and gene expression in plant.

### Claim 6

A method for transforming a plant cell, comprising:
providing a vector comprising: SEQ ID NO: 1, a set of ordered unique restriction endonuclease cleavage sites for insertion of heterologous DNA, and a heterologous DNA encoding a gene product inserted into the unique restriction site; and
introducing said vector into the plant cell.

### Claim 7 (depends on 6)

The method of claim 6, wherein the RB has a 208 base pair sequence and the LB has a 155 base pair sequence.

### Claim 8 (depends on 6)

The method of claim 6, further comprising regenerating a transgenic plant from the transformed plant cell.

### Claim 9 (depends on 6)

The method of claim 6, further comprising expression of the heterologous DNA in the plant cell to produce gene product encoded by the heterologous DNA in the plant cell.

### Claim 10 (depends on 6)

The method of claim 6, wherein the vector further comprises a selection marker for introduction of the heterologous DNA into the plant cell, the selection market located between the LB and RB sequences.