Volterra/San G + Heart of Tuscany + Assisi + Umbria Itinerary

Jie Bo:
September/October is a wonderful time to be in Tuscany. If you think you can manage, I would recommend a rental car. If you pick the car up in Siena you can drive through Tuscany and on to Orvieto primarily on 2 lane roads, which makes the switch to the opposite side of the road easier. (TIP: The driver is always in the middle of the road and the passenger on to outside. This is particularly helpful when navigating a traffic circle.)
Since you have only 1-2 nights, it would be better to head south from Siena and visit Pienza and Montepuciano before continuing south to Orvieto.

The car can be returned in Orvieto and you can continue to Rome via train.
For accommodations a hotel would be best for such a short period. Perhaps on day one you explore Tuscany and spend 1 night in Montepuciano and then drive on to Orvieto and spend one night before catching the train to Rome. We stayed in Hotel Duomo in Orvieto and the accommodations were great.
I hope you have a wonderful time.

Siena and Montepulciano are the two Tuscan towns you want to visit. From Florence take a morning bus to Siena (1h 15m) and sleep there for two nights. The Albergo Bernini https://www.albergobernini.com/en/ is where I stayed in the old town.
The next day take a bus to Siena’s train station and a direct train to Montepulciano (get an early start, the only morning train I see departs at 8:13a) taking 1h 15m and a bus from Montepulciano’s train station to the town center (30m).
Orvieto is an easy day trip from Rome’s Termini station (1h 15) and you'll need to take the funicular to the top of the town: https://www.trenitalia.com/en.html

Thank you both for your sensible recommendations. I think we'll pick up the rental car in the morning at Siena and depart towards Montepulciano before spending a night there. We'll then depart towards Orvieto the next morning, drop off the car and spend one night there. Following on with an easy train from Orvieto down to Rome.
Once again, thank you for responding so quickly!

Jie Bo,
When you rent a car in Siena, get on the Val d’Orcia (SR2) road. To get to Montepulciano you’ll need to turn left onto SP146: https://lovefromtuscany.com/scenic-drives-in-tuscany/; this is why you go to Tuscany. No need to get an early start. My first 30 minutes on this hilly windy very scenic road was covered with fog and I started out late morning. The drive takes 1h 30m if you don't stop along the way.

The first thing you mentioned in your post was that you wanted to avoid driving if possible, and that is completely doable with both of your itineraries. Italy has trains and buses going everywhere. It would, however, be more practical (instead of Volterra/San G) to take the train from Florence to Orvieto and the bus to Civita. Or a long day trip by train from Orvieto to Assisi. You can also rent a car in Orvieto, and then take the train to Rome.

Jie Bo,

We too will be doing the Heart of Tuscany drive next fall. We are Americans that just spent a few weeks driving in Australia on the opposite side and, although the learning curve is steep getting out of the city, it wasn't too hard to adjust to driving.

I highly recommend trying to rent your car from your home country. For Americans the price is less than half then going through the same rental car companies in Italy. We're going to pick up our car outside of Siena, take 3 days to see Tuscany, and then return it in Napoli.

We are still working on our accommodations, and so far in the bigger cities (Firenze) we are staying in hotels but in the smaller cities (Orvieto) we are opting for AirBnBs.