The King made a quip about living "long enough" to see a tree he planted grow on the final day of his Italian state visit.
Charles, 76, cracked the joke as he met representatives of the Circular Bioeconomy Alliance (CBA) to hear more about their work in relation to biodiversity, climate and human health.
As part of his engagement, the monarch also planted a tree in the gardens of Villa Wolkonsky in Rome, donated by President Mattarella from the Castelporziano Estate.
After performing his personal ritual of waving the spade in the air once he had shovelled soil around the roots, the King turned to the British embassy staff gathered on the lawns.
He quipped: "Well I do hope that I shall live long enough to see a little bit of growth in the tree."
As the assembled crowd clapped and cheered, Charles, who is receiving ongoing treatment for cancer, said "one more," in reference to the sapling.
The King then unveiled a plaque to commemorate the occasion, before pointing at the newly planted tree and saying: "Needs a bit of water."
Historic visit
The King and Queen have had a packed schedule of engagements since beginning their four-day state visit to Italy on Monday, which also coincided with the couple's 20th wedding anniversary.
New portraits of Charles and Camilla were released on Monday to commemorate the occasion, with the couple attending a state banquet on their actual anniversary on 9 April.
Their Majesties were guests of honour at the black-tie event hosted by Italy's President Sergio Mattarella, where the King jokingly referred to the banquet as a "small romantic, candle-lit dinner for two".
Meeting with the Pope
Charles and Camilla also privately met with Pope Francis on Wednesday.
The pair visited the recuperating pontiff at the Vatican, with Buckingham Palace releasing a photograph.
In the photograph, the Queen is shaking the hand of Pope Francis, who is seated, while the King stands next to her smiling.
There was an exchange of gifts between the couple and the head of the Roman Catholic Church, and Charles can be seen holding a small red box.
The King and Queen concluded the final day of their trip in Ravenna on Thursday.