Always use strict and warnings.  With that, you'd have gotten warnings like Variable "$open" will not stay shared, which might have been a hint that there's still something wrong with the scoping of the variable $open.

The problem is in particular that you've declared $open locally to the callback function push_button, and that you've declared the other callback functions locally to that function (which I had only realized after having indented your code reasonably...).   Perl does not have local subroutines, and if you declare named subs within other subs, they're essentially parsed as if they were declared top-level. Hence the scope issue.

Here's a stripped down version (with "minor" edits to make it actually runnable...), which uses real global variables for $open and $te (for which the same issues apply), so that they can be shared across various callback routines.

#!/usr/bin/perl -w
use strict;
use Tk;

my $open;
my $te;

# Entry Window
my $mw = new MainWindow;

# The START button:
my $button = $mw -> Button(-text => "Start",-command=>\&push_button)->
+pack();

MainLoop;

sub push_button {
    my $mw = new MainWindow;
    $mw -> geometry ("1200x600");

    # Menu bar
    
    $mw->configure(-menu => my $menubar = $mw->Menu,
                           -background => 'white');
    my $file = $menubar->cascade(-label => '~File');
    my $edit = $menubar->cascade(-label => '~Edit');
    my $help = $menubar->cascade(-label => '~Help');
    
    $mw-> Button (-text =>'Open', -command =>\&open_file)->place(-x=>2
+40, -y=>35); 
    $mw-> Button (-text =>'Get Statistics', -command =>\&get_statistic
+s)->place(-x=>320, -y=>35); 
    
    $te = $mw->Scrolled('TextUndo')->place(-x=>240, -y=>70);

    MainLoop;
}

sub open_file {
    $open = $mw->getOpenFile(
                #-filetypes => $types_OPEN,
                -defaultextension => '.sff'
            );
    $te->Load($open);
}

sub get_statistics { 
    my $fastafile= $open;
    print STDERR "selected file: $fastafile\n";
}
[download]

A few more comments:

• Indenting your code in some sane way already gets you a long way to better understanding it.
• Always try to provide code samples that actually run. That way you significantly increase the chances of people looking into your problem. In fact, I only went to the trouble of editing your sample code because I just happened to have a few idle minutes.
• I think (but I'm no Tk expert) that having another MainLoop within a callback function is somewhat "unusual" style.
• And, at the risk of repeating myself: use strictures/warnings — they have been invented to help you.

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