Somatic Fusions in Your Kitchen

This is callus from Hylocereus undatus, Vietnamese white dragon fruit. I don't know if it's embryogenic or how well it will release protoplasts.

This is callus from Hylocereus undatus, Vietnamese white dragon fruit. I don’t know if it’s embryogenic or how well it will release protoplasts.

I recently got a comment on my blog regarding somatic hybridization and whether or not you could do it at home. Yes, I suppose you could, but it would require some specialized equipment and a lot of time. The information on how to make somatic hybrids is publicly available, in published scientific journals; You may need to go to a university library to get it, but the information is available.

Let’s start at the beginning: What is a somatic fusion? Somatic fusions are the combination of two somatic cells, which is used to differentiate the process by which normal sexual hybrids are made, i.e. sexual cells. Somatic cells are the adult cells which contain the full complement of DNA, unlike sexual cells which contain only half (i.e. half from mom, half from dad). Somatic cells can’t usually be combined unless we make them, using either chemicals or electricity. Plant and animal cells can be combined, but they don’t usually live very long. It could also be done to fuse an elephant with a rhinoceros, in order to make the famed “elefino”, but I’ve never tried it; I deal mostly with plants. In order to fuse somatic cells in plants, first the cellular wall must be removed. This can be done mechanically, by using osmotic pressure to get the cells to contract away from the cellular wall and then chopping them open, or by using enzymes to remove the cell wall, leaving only the naked protoplast. The latter is the method more commonly used. Enzymes used are typically cellulase or Macerozyme R-10, which is a combination of several enzymes, available from Phytotechlab.com (http://www.phytotechlab.com/detail.aspx?ID=432).

This is some Citrus protoplasts. The green ones are derived from leaves while the clear ones are from callus in suspension. In the middle you can see a fusion, that is mostly clear and part green.

These are some Citrus protoplasts. The green ones are derived from leaves while the clear ones are from callus in suspension. In the middle you can see a fusion, that is mostly clear and part green.

A protoplast is a naked cell, and once stripped it can be fused with another protoplast, either chemically (using a chemical called polyethylene glycol, usually abbreviated P.E.G.) or with electricity. I’ve never done electrofusion, as it requires some expensive, specialized equipment which we didn’t have in our lab. (Although we recently got it, but I haven’t tried it yet.) PEG is commonly available, as it is used as a laxative (Miralax). I don’t know how much it varies from the chemically pure PEG for laboratory use, but it is commonly available. We use PEG 8,000 from Sigma, where the 8,000 refers to the molecular weight. (It comes in a number of molecular weights; some of the scientists in our lab found that 8,000 works the best with the highest number of fusions per application, but this is unpublished data.)

Once you have the isolated protoplasts, then apply the PEG, and very carefully wash it off, then you have to wait and culture it for a long time (feed and/or transfer to new plates every two weeks). The fusions I did last July, are now only regenerating into plants (March), so that’s 8 months. So another ingredient: patience.

I guess there is another question I haven’t answered: Why make somatic fusions? The main reason is because the cross is either impossible or highly difficult otherwise. In citrus, a common trait is apomicty, where the seed you plant is a clone of the mother. Even if you make a cross, the seed is a clone, so it becomes a genetic dead end. Most of the varieties of citrus have arisen through mutations for that reason (Ruby red grapefruit is a mutation of a mutation of a mutation of the original seedy, white Duncan grapefruit). However, if you fuse a sweet orange with a pummelo, you can make something genetically similar to a grapefruit. The other reason is that unlike the genetic gamble of a sexual cross, in a fusion you have both complete genomes of each parent.

After making the fusions, and transferring them every two weeks nigh unto infinity, you begin to get plants. This is my first hybrid; you can tell because it has trifoliate leaves, a characteristic of the leaf donor (which is non-embryogenic).

After making the fusions, and transferring them every two weeks nigh unto infinity, you begin to get plants. This is my first hybrid; you can tell because it has trifoliate leaves, a characteristic of the leaf donor (which is non-embryogenic).

If you want to add disease resistance, this is much simpler than crossing the two and growing out a large population and screening for the few that inherited it plus desirable fruiting characteristics. Citrus also has long juvenility, from 5 to twenty years from seed until fruiting, which as a breeder makes your life difficult. The wider the cross, though, the less likely you will have a fertile cross. My boss made fusions of box orange (a Citrus relative that is graft compatible, but not  sexually compatible with Citrus (Ok, someone did make a cross in Brazil, but it must not have ever flowered, because there was no subsequent paper)) with Citrus in the late 80’s early 90’s that have never flowered. 25 years is a long time to wait and not get results. He’s got a Nova + Citropsis hybrid that may yet fruit, but we’ll see. (Fusions are also labeled with a + and not an x, since it is additive).

Let’s get to the meat of this: Could I do this at home? Let me say maybe, if you’re willing to purchase or make the equipment (you can often find used lab equipment inexpensively… Well, compared to buying it new). Here’s what you will need:
Laminar flow hood (essential to maintaining a sterile environment to make your fusions, here’s a link to build your own: http://www.fungifun.org/English/Flowhood)
Centrifuge with a swinging bucket (These can be found on eBay, Without a rotor they can be fairly cheap, but you will want a rotor)
Sterile petri dishes (6 cm)
Sterilizing filters, with a vacuum pump
Sterile 9″ glass pipettes, and pipette bulbs
Orbital Shaker (Used ones can be found on eBay)
Scalpel with sterile blades (for feathering leaves)
(Blades can be purchased sterile, but it also helps to have a way to sterilize it, like 95% alcohol, glass bead sterilizer, or some other sterilizer)
Sterile plant material (either from callus or leaves)
PEG
Reverse osmosis water
Various feeding mixtures, like MS media with sugars, see papers you reference
40µ filter
15 mL Centrifuge tubes
Parafilm, laboratory sealing film
Autoclave or something similar (Like a pressure cooker)
Flow cytometer (optional, you could count chromosomes with microscope or send your samples to a lab)

This shows the little plates with callus (from suspension) in enzymes, and feathered leaves in enzymes. Callus is Meiwa kumquat and leaves are from Ray Ruby grapefruit. This is part of my PhD project.

This shows the little plates with callus (from suspension) in enzymes, and feathered leaves in enzymes. Callus is Meiwa kumquat and leaves are from Ray Ruby grapefruit. This is part of my PhD project.

To make fusions, it’s a two day process. Here it is abbreviated:
Day 1: Sterilize leaves (if needed) and feather them (use scalpel to cut the leaves into many strips). Insert the leaves into enzyme mixture and vacuum for 15 minutes (to get the enzymes into the tissue). Place on shaker overnight, at 25 rpm, at room temp. Also add enzymes to callus, incubate on shaker overnight. (Citrus requires embryogenic callus, and then I use non-embryogenic leaves for the other half, which will not regenerate on their own).

Day 2:
Filter the protoplasts out of the enzyme mixture, wash using the centrifuge (800rpm, 10 min. several times) Place the washed protoplasts in equal amounts and centrifuge, then resuspend and put a couple drops per plate and add a couple drops PEG per plate. After 10 min, carefully add some high pH stuff, then another 10 min, begin washing by sucking away the liquid carefully and adding the feeding mixture. Do this several times. Seal plates, Then place in a temperature stable place, around 70 degrees F, in the dark for two weeks.

In a day or so, the protoplasts will regenerate their cell wall, and will lose any green color from the leaf protoplasts. This can be observed under a microscope, but the best microscope to use is an inverted microscope, which can look through the bottom of plates.

Here is a portion of my plates, that I have to transfer to new plates every two weeks. Once embryos form roots or shoots I transfer them to the boxes, after which I will test them on the flow cytometer.

Here is a portion of my plates, that I have to transfer to new plates every two weeks. Once embryos form roots or shoots I transfer them to the boxes, after which I will test them on the flow cytometer.

After 2 weeks, add 10 drops of solution to change the osmotic pressure. Then in two more weeks, if colonies are growing, then transfer them to a nutrient agar plate. Then transfer them every two weeks until plants begin to regenerate. At first you will get little green pro-embryos, which then turn into weird green blobs, which may eventually make a root or a shoot. Then out of the hundreds of plants you regenerate, test the leaves using a flow cytometer, to identify the tetraploid hybrid. Once you have the hybrid you can stop looking if you want, because in somatic hybrids there is very little genetic variation for the same cross. The only variation will arise by mutation, which can and does happen.

And there you have it! That’s how you can make your own somatic hybrid at home.

As you have probably already come to the conclusion to by now, will it’s not impossible to do at home, it is unlikely that you will. It will be expensive to set up, requires specialized equipment, and most importantly, a dedicated, clean (sterile) environment. The laminar flow hood is the most important piece of equipment, along with good sterile technique; I get contaminated cultures all the time, it’s just impossible to eliminate all of the fungi and bacteria that are floating around, and every time you open a dish to feed it or transfer, you are creating an opportunity for them to enter. The worst part is the amount of time involved; I do this all day on a daily basis. You on the other hand probably have a job and it’s probably not this.

If you want to learn more about how to do this, there are some books available:
Plant Cell and Tissue Culture, by Indra K. Vasil and Trevor A. Thorpe. Pub 1994.

Most information, especially regarding the species you want to try, will be found in scientific papers. Search for ‘protoplast’ or ‘somatic fusion’.

If you want to learn about doing tissue culture at home, check out http://www.kitchenculturekit.com.

Here’s a video I made about somatic fusions: http://youtu.be/RBqxRPujZfo

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6 thoughts on “Somatic Fusions in Your Kitchen

  1. Thanks for your response. I didn’t see your latest entry until today. One of the links to your pictures is broken, it only contains a comment. You are engaged in exciting work.

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