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So I was recently taught cloning in plants and I came to wonder what is the need to first put the gene of interest in the entry vector plasmid and then the final vector plasmid before finally transforming agrobacterium? Can someone make it make sense to me?

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    $\begingroup$ Welcome to the site. Please visit the help center and take a tour. If you could explain (edit into your question) which cloning procedure you are using in detail it would help us to know exactly which methodology you are using. In addition, this site has a requirement of evidence that you have had a go at answering this question for yourself, otherwise it will be closed as homework. $\endgroup$
    – bob1
    May 24 at 9:02
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    $\begingroup$ There are a lot of different vector systems and some have 2 components, so including the details for your vectors would help. As bob1 notes, reading the material for that system might allow you to answer the question yourself. $\endgroup$
    – Michael_A
    May 24 at 9:43

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If you mean the binary system using the Ti plasmid:

The main issue is that on the massive Ti plasmid there is a difficulty locating unique restriction sites (see section "Introduction of Genes into Plants by Using Agrobacterium"):

However, Ti plasmids are very large and T-DNA regions do not generally contain unique restriction endonuclease sites not found elsewhere on the Ti plasmid. Therefore, one cannot simply clone a gene of interest into the T-region. Scientists therefore developed a number of strategies to introduce foreign genes into the T-DNA. These strategies involved two different approaches: cloning the gene, by indirect means, into the Ti plasmid such that the new gene was in cis with the virulence genes on the same plasmid, or cloning the gene into a T-region that was on a separate replicon from the vir genes (T-DNA binary vectors).

Both of these approaches mentioned involve a two-step cloning procedure for using the entry vector to add the payload to the Ti plasmid.

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    $\begingroup$ Edited to link ref and add quote. $\endgroup$ 2 days ago

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