Scottish Conservative leader Ruth Davidson has said she is greatly disappointed in her former Better Together stablemates for allowing nationalists to join their parties.
Labour and the Liberal Democrats have signalled they would permit their members to campaign for independence, despite siding with Tories in the campaign to keep Scotland in the United Kingdom.
In a speech in Stirling, in the shadow of the Wallace Monument, Ms Davidson called Labour leader Kezia Dugdale "weak" and urged her to reverse her party's "chaotic position".
Ms Davidson defended her decision to keep the union at the centre of her campaign, insisting the threat of independence has not receded and will be top of the SNP's agenda if they win the Scottish election in May as polls predict.
She told the Press Association: "Having worked with both Labour and the Liberal Democrats quite well over Better Together, I'm really disappointed in them for changing and softening their stance on the United Kingdom.
"Allowing their candidates and future MSPs to campaign for independence says to me that they care more in this election about getting back what they have lost than protecting the decision that they helped win for the country. That disappoints me greatly."
She said her party's internal polling is "very encouraging" and has given candidates "a spring in our step" in the race to beat Labour into second place.
In her speech, she accused Labour of siding with the SNP too often, insisting Scotland "has had enough of Labour's nodding dog" and must turn to "a Tory terrier".
She attacked Labour's "useless" attempts to "smear" her party, describing them as "one part negativity to two parts total incompetence".
She said: "Do we really have to suffer five more years of this third-rate party, blundering about, punching wildly, and never landing a blow?"
She added: "We are being attacked by Labour and the Lib Dems for putting the issue of the union at the centre of our campaign.
"Their point appears to be that we can defeat independence by simply wishing hard enough - that if we ignore it, it will go away.
"Hence the reason why Labour and the Lib Dems both say they are happy for their candidates at this election to back independence if they wish. To have future MSPs campaign for the break up of Britain.
"I just think they've got this one wrong.
"I would like nothing more than the threat of independence to go away... but the plain fact is that's not enough. Not nearly."
She said independence will be "first, last and always at the top of the SNP's in-tray on May 6".
"I call on the Labour Party leader (Kezia Dugdale) today to reverse her chaotic position on this," she said. "Her position right now is that Labour candidates are free to do as they wish. I think that's weak.
"I think she should make it clear: that under her, no Labour candidates will be able to campaign for independence. This is too important. She needs to take control."
With the UK Conservatives currently divided between Eurosceptics and EU supporters, Ms Dugdale insists there is no room within her party for similar divisions on the UK.
She told the Press Association: "This is an article of faith for us.
"Nobody thinks the EU is the same as the nation state of the United Kingdom.
"We have been an integral part of the United Kingdom for over 300 years.
"We have never said we're going to throw people out or anything like that, but we do require our candidates and MSPs to believe in the tenets and policy positions of our party.
"I'm not sure we've got that many applications from nationalists, but we think our candidate vetting procedure is very robust and if people are huge independence campaigners that we might spot them and ask whether it is really the Conservative and Unionist Party that they want to be a candidate for."
A Labour spokesman said: "The Tories must think Scots zip up the back.
"They've been claiming to be in a 'taxpayer's alliance' with the SNP and failed to oppose the SNP Budget, which will mean hundreds of millions of pounds of cuts to schools and local services across Scotland.
"Faced with the choice between SNP cuts or using the powers of the Scottish Parliament, we choose to use the powers and set the Scottish rate of income tax 1 penny higher than the rate set by (Chancellor) George Osborne to stop the cuts."
Scottish Liberal Democrat leader Willie Rennie said: "Today I visited student nurses in Edinburgh to observe and take part in nurse training.
"Over the past eight years our NHS has been put under more and more strain while the focus has been on independence.
"Meanwhile what has Ruth done? She's spent the day dragging out the constitution debate. Again.
"That shows exactly where her priorities lie. When will she have something to say about the issues that affect Scots' day-to-day lives?"
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