More than 130 business people in sectors ranging from finance to whisky have signed an open letter arguing that the business case for Scottish independence “has not been made”.

The letter, organised by Keith Cochrane, chief executive of Weir Group and signed by figures included Ian Marchant, former chief executive of the utility SSE and Andrew Mackenzie, chief executive of miner BHP Billiton, is the biggest business intervention in the referendum debate so far.

It offers a boost to the campaign against Scottish independence after Alistair Darling, former Labour chancellor and leader of the pro-union Better Together group, was widely seen as losing a televised debate with Alex Salmond, Scotland’s first minister.

The letter, published in The Scotsman newspaper on Wednesday, says the business leaders, including Ian Angus MacKenzie, chief executive of Harris Tweed Hebrides and Niall Booker, chief executive officer of the Co-operative Bank, have “looked carefully” at the arguments for, and against, independence.

“Uncertainty surrounds a number of vital issues including currency, regulation, tax, pensions, EU membership and support for our exports around the world; and uncertainty is bad for business,” it says.

“Our conclusion is that the business case for independence has not been made.”

Pro-union politicians have repeatedly complained that business leaders have been too reluctant to enter the business debate. Better Together’s attempts to organise a group of business people have had little impact.

The pro-independence Business for Scotland campaign group responded to the letter organised by Mr Cochrane by saying its 2,500 members had concluded that neglect from London government and poor UK economic policy meant their best interests lay in Scotland becoming an independent country.

"The biggest business concern we have . . . is the substantial and growing antipathy towards the EU at Westminster,” said Tony Banks, who chairs Business for Scotland.

With just over three weeks until polling day, Scotland’s nationalist first minister Mr Salmond won the televised debate convincingly, crowning a confident performance with an appeal to his countrymen: “This is our time, our moment: let us do it now.”

When David Cameron returns from Cornwall to 10 Downing Street on Wednesday, the prime minister will huddle with advisers to assess whether Mr Salmond can win the independence vote on September 18.

The verdict of those closest to the prime minister is that a Yes vote remains highly unlikely: the polls continue to give the No side a double-digit lead. An FT tracker poll puts the margin at about 13 percentage points.

But Mr Cameron’s team says there is no room for complacency. Aides concede that Mr Salmond has injected new dynamism into the nationalist campaign but insist that the first minister has left too many questions unanswered.

“Following last night’s [Monday’s] debate the prime minister concluded that the first minister still has no answers on currency or how to fund public services while relying on volatile and declining oil revenues,” said a spokesman for Mr Cameron.

The prime minister is expected to travel to Scotland shortly to try to inject a more optimistic message into a pro-union campaign that has focused heavily on dire warnings of the economic dangers of independence.

“He will do the sunny uplands message,” a supporter said. “He’ll send the message that the rest of the UK wants Scotland to stay.”

But the No campaign has no intention of easing up on the negative campaign

Mr Darling repeatedly warned in Monday’s BBC debate about the uncertainty over what currency an independent Scotland would use.

A snap ICM/Guardian poll suggested that Mr Salmond won the debate by a margin of 71 to 29, prompting recriminations from the Darling camp over the way the BBC staged the debate, which turned into a shouting match.

“BBC Scotland is a disgrace,” said a senior member of the Better Together campaign, who claimed the debate should have been refereed more effectively and that the audience seemed to be disproportionately nationalist.

Blair Jenkins, chief executive of the Yes campaign, said Mr Salmond had the better arguments and Mr Darling had been left “floundering”.

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But he admitted that the first minister’s debating triumph might not immediately show up in the polls.

As the campaign enters its final leg, Mr Salmond’s campaign will focus on his claim that the only way to protect the National Health Service and stop welfare cuts is to ensure that the people of Scotland always get the government they vote for.

Mr Salmond also challenged Mr Cameron to a debate on Scottish independence.

“Let’s have the real leader of the No campaign, David Cameron. Let’s have him in Scotland now, let’s see if he can do any better than Alistair Darling did,” he said.

The pro-union side will continue to stress the “wallet issues” identified by pollsters as being the key areas concerning undecided voters, notably women and those from poorer backgrounds living in the urban west of Scotland.

Mr Salmond’s promise of a confident, progressive Scotland forging its own path in the world clearly had a stronger emotional connection with voters on Monday: Mr Cameron still believes the No campaign can win the battle for their heads.

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