SS: Welcome, Bill. Thank you for joining me today.

WGK: I’m happy to be here. Thank you. 

SS: Could you please explain what is plant tissue culture and what role it plays in crop improvement?

WGK: Sure. Tissue culture is simply starting with small explants from various plant species and, growing them in vitro for various purposes. The technique has been widely used for many years for plant propagation and micropropagation of many horticultural crops, many crops that are difficult to reproduce sexually. It is a very integral part of many double haploid protocols which are widely used in agriculture today for accelerated genetics. And of course, it provides the platform for transformation and editing protocols for many species.

SS: Is there any example for general audience—one or two examples—where it is very useful for the society in the last 30 years?

WGK: When you look at asexually propagated agricultural crops like sugarcane, it has been extremely valuable. In recent days in the field of editing where they can’t, for example produce, large scale immature embryos for transformation. And they must rely on different ex-plants and different techniques of doing their culture for editing purposes. And that’s been extremely successful. And I think the probably, some of the major agricultural crops, such as wheat and maize, double haploid production relies on in vitro embryo rescue to accelerate the process and identify haploid plants and then take them through the doubling process,

typically, in vitro, so that they don’t have to, to use these harsh chemicals for chromosome doubling in the field, for example.

SS: Your research is focused on improving transformation and regeneration of cereal crops.

And some of these crops are extremely difficult to either transformation or regeneration.

How does your work help to solve that bottleneck?

WGK: When I started working in maize tissue culture, transformation and regeneration were extremely difficult. Improvements in tissue culture techniques traditionally had been done through media manipulation. Through very methodical and painstaking testing, for example of media grids and hormone grids to find which combinations of hormones and salts produced the best tissue culture response for a genotype. 

In maize when I started back in the 80s, there was only one genotype that was amenable to efficient tissue culture. And that presented a good target for transformation. That was a hybrid between B73, which is stiff-stalk inbred, and A1-88, which is agronomically poor inbred, but an inbred that provided unique tissue culture properties and very high somatic embryogenesis rates.

It was very easy to manipulate and culture. That became the first target that everyone really focused on for transformation research in corn. 

That type of media, tissue culture research, was really pioneered by some excellent scientist. Murashige and Skoog two of the pioneers in that area that really kind of established the techniques for the field and industry. I had to do a lot of that type of research myself, but I didn’t have the patience for it, honestly. I’ve always liked a quote by a science fiction writer Robert Heinlein who said that “progress isn’t made by early risers. It’s made by lazy men who want to find easier ways to do things.” And I think that probably sums up my research career.

SS: Could you please explain what are the Dev Genes or Morphogenic genes and how can someone use them to improve plant transformation and regeneration?

WGK: As the name implies these are genes that are very critical at the different transitional stages during development of the plant that my lab is focused on. Well, the first one that we focused on was LEC1 which is a gene involved in embryogenesis. And then later we moved on to WUSCHEL and BABY BOOM. We tested these individually and we started testing them in combinations. And we found that WUSCHEL and BABY BOOM together were very effective at the point that I started using it.

BABY BOOM was discovered by an excellent developmental biologist by the name of Kim Boutilier. She discovered BABY BOOM studying microspore embryogenesis in brassica and looking at differential expression between embryogenic and nonembryogenic responses. And BABY BOOM jumped out as being one of the genes involved in very early triggering of embryo formation.

WUSCHEL has been known for quite a while as a gene that was essential for maintenance of meristem structure. It is kind of the central gene that signals and controls such things as meristem size. BABY BOOM wasn’t a surprise in terms of being able to use it for embryogenesis. WUSCHEL for me turned out to be more surprising because I expected the ectopic expression of WUSCHEL would produce de novo shoots. And in many crops, it was found that a pulse of WUSCHEL expression will actually stimulate embryo formation.

Extremely exciting, when we first saw it.

Those aren’t the only genes now that are used in this manner. Researchers at University of California, Davis, have done some fantastic work with the GRF/GIF and in combination.

SS: Yeah. I think recently in Plant Journal, Lauren Pauwels group published GRF/GIF in maize. right? 

WGK: Yes, Lauren Pauwels group at Vlaams Institute for Biotechnology at Ghent have done some great work using these types of genes.

WOX5 has been published as stimulating, improving transformation in wheat and other crops and recently WOX2A has been found to stimulate somatic embryogenesis in maize. I think there are going to be more genes like this that are going to be useful. And it’s just a matter of people being creative and really studying the developmental biology literature and picking and choosing. What they think are going to be good candidates and giving them a try.

SS: Right, I think you answer my fourth question—about the idea how WUSCHEL and BABY BOOM came into this scenario. Would you like to add something more?

WGK: As I mentioned, you know, continuing to fiddle around with tissue culture media wasn’t my inclination. It was necessary. We paid close attention to it. But I wanted to try to find alternatives. And in the beginning, when we were trying to work on transformation finding good selectable markers was difficult. So, we were testing out a lot of herbicides and antibiotics, kind of, you know, the types of resistance genes that you would intuitively turn to. 

But I became interested in the idea of trying to find plant genes that would stimulate growth. And at the time, cell cycle cancer research was just booming in the mammalian field and in human research area. So, we started cloning maize cell cycle genes, and we cloned all the maize cell cycle genes we could and tried manipulating them in different ways to stimulate cell division. And we found that the cyclin D and REP A were very effective at stimulating growth above and beyond normal tissue culture rates. S0, we could use that as a positive selection.

But it didn’t quite have the power to get us into more recalcitrant genotypes. So, it worked in things that were already cultural. But in these difficult inbred such as B73 it wasn’t effective.

That made us start looking in other directions and morphogenetic genes. Just immediately when we started seeing these first reports from Brian Miki lab in Ottawa with Kim Boutilier, and John Harada lab at UC Davis had some wonderful papers that came out on several genes. I mean it was just too enticing. We jumped on that area as quickly as we could. 

SS: To that point—you started just one inbred line of the maize which is amendable to do tissue culture but how many lines you can do at this moment?

WGK: When we started working with WUSCHEL and BABY BOOM we were using a week promoter for WUSCHEL and then a strong constitutive promoter for BABY BOOM. And with that combination, we found that we could transform about 20% of the inbreds that we tested.

That was a good start. But then we were always looking for new promoters, and we started concentrating on trying to find promoters that were very highly expressed in the scutellum of the immature embryo. And we were lucky enough to work with a fantastic bioinformatician here at Corteva by the name of Mauricio La Rota, who found the PLTP gene for us through a lot of filtering and sifting through transcriptome data in different genotypes. That PLTP promoter we found gave us initial expression levels that were about four to five-fold stronger than the ubiquitin promoter. And that turned off after four or five days. It just provided a strong pulse.

Then we also switched to an auxin induced promoter for WUSCHEL. So, with the auxin levels that we typically use in our culture media, those provided a strong pulse of WUSCHEL and BABY BOOM. We found that switching with those promoters then would allow us to be starting with immature embryos, transform any inbred that we tested in maize. Still the frequencies varied substantially.

Some embryos would give us high transformation efficiencies, and some were still low, but they were all transformable. That gave us the flexibility then to start working on genome editing across all our germplasm. And surprisingly, it also worked in all the sorghum varieties that we tested. And it worked extremely well for wheat. So, it was a very useful combination.

SS: I’m seeing in the literature—people are also using this system for many other crops.

WGK: Yes. Then with the PLTP promoter, the other very exciting variation on this theme that we found was that if we took three viral enhancers and placed them in front of the PLTP promoter and now use that promoter to drive WUSCHEL expression, that it produced so much WUSCHEL in the transgenic cell that cell was no longer regenerative. Cells were pumping out so much WUSCHEL protein. WUSCHEL is a diffuse protein, it would move into the surrounding the neighboring cells and stimulate somatic embryogenesis in its neighbors. That is an extremely useful method.

And we started then using two agrobacteria for our transformation, one to deliver WUSCHEL and one to deliver our trait genes or our editing genes. We could vary the concentrations of agrobacteria to skew the outcomes one way or the other, depending on what we were after. I think that’s, that’s going to have a lot of utility in the future for cereal transformation.

Then finally, most recently, we’ve continued to alter the promoters on these genes. And we’ve developed leaf transformation, which I’m most excited about. Because it’s producing a method that is going to be much more user friendly and much easier for labs without sophisticated machinery and without the infrastructure to grow donor plants to produce immature embryos. So now you can just grow seedlings in your lab and transform various cereal crops, which I think is going to be extremely useful and make cereal transformation hopefully much more egalitarian, democratize the method for use across many more academic labs. We still have a lot of work to do in that area, but it’s going well.

SS: Two months back I spoke to Steven Strauss from Oregon State. He mentioned he is using altruistic methods in his laboratory.

WGK: Yes. His lab is really doing some fantastic research in poplar and eucalyptus.That’ll be really exciting to see that these developments. 

SS: Do have any hypotheses on how do WUSCHEL and BBM change the cell fate? How much it’s genetic versus epigenetic? How do they affect epigenetic landscape and the cellular clock?

I’m reading some of the very old literature like Alan Turing’s Chemical basis of morphogenesis.

Now we understand these are the hormones which are the morphogenetic factor. Then Waddington landscape. Now we can take the cell back from differentiated cell to undifferentiated cells—we can go back in the landscape. I’m very curious how do WUSCHEL and BABY BOOM affect epigenetic landscape or genetic architecture?

WGK: That’s an exciting question. Based on what we know from the literature the genome architecture, the epigenetic landscape, when you compare an immature embryo, and a leaf cell must be radically different from each other. There has been some nice work looking at epigenetic changes that occur during development. For example, in maize methylation status around the baby boom locus changes during development. Some of the basic and the biological framework is known. It’s just a matter of someone doing the careful experiments on how these changes occur during transformation.

Working in industry, we must remain focused on practical applications. So, I just as much as wanted to dig into those areas, we just haven’t had the bandwidth to do it. It is an exciting area that I hope someone is going to start exploring and really provide some of the answers that you’re asking about, because all we can do right now is speculate. 

SS: All the cells have the same DNA, right? Whether it is a somatic embryo or callus or whatever tissue, everything has the same DNA. But why they are so much different?

WGK: Yes. It’s a fascinating area. Another kind of practical area that I’ve been involved in for many years is targeted integration, first through using site specific recombinases such as CRE and FLP system. And then of course, more recently using mega nucleases or Cas9. One of the things that we know from our work is that some genomic locations that we’re interested in targeting are more recombinogenic. They’re more accessible for integration either through FLP mediated integration or through homologous recombination.

That mean most likely that’s due to changes in accessibility at that location during development. Whether you have chromatin condensation going on or the area is more relaxed and active at that time. One of the avenues that I think is still going to be interesting to try to explore is—how to relax an integration locus for efficient targeting. I think it’s probably kind of fun.

SS: Yeah, I think there are recently at least three publications, one from Danforth Center. They have used recombinase and transposase and engineered those for the plant. And there are two publications from Arch Institution. They did the same thing in the animal system. It is a programable system. So, it can be programmed and then they can do whatever you want to do on the genome.

WGK: Yeah. Very exciting–Really cool work.

SS: Yeah. So, coming to another question, which is maybe interesting for our audience. What are the impractical scientific ideas—most of the people believe at time that these are impossible to do—but only few people believe and pursue these ideas. So, what are those ideas in plant science or in agtech that become practical in your 40-year career.

WGK: I think we’ve got a couple examples. The first is efficient targeted integration through homologous recombination. Thirty years ago, we we’ve always looked to the mammalian literature to kind of guide us and give us an idea of what to expect in plants and in higher eukaryotes. Homologous recombination frequencies for people that have been working on trying to do targeted integration have been extremely low—up to 1 in 1000 type of event. Probably the person who’s had the most experience in this area over the years is Holger Puchta lab—who has just done brilliant work over the years, really pioneered that area. All of his early publications in Arabidopsis, the homologous recombination frequencies were, were very low.

One pleasant surprise—tools have improved dramatically with the advent of the Cas system. Cas make targeting much more flexible, but it inherently shouldn’t change the recombination frequencies. Whether a cell is going to predominantly repair through non-homologous enjoining or homologous recombination set by the cellular environment, and you can’t change that. We looked at mammalian systems and it’s clear in the mammalian literature that, if you have cells that are stuck in G1 phase of the cell cycle, that non-homologous end joining is going to predominate, and the cells that cycling more rapidly through S phase and G2, you’re going to increase your HR frequencies.

Here we got lucky because one of the dramatic impacts of WUSCHEL and BABY BOOM at the high rates that were expressing these genes are, cycling time in these small somatic embryos is about 10 to 12 hours. The cells are just dividing rapidly, and it provides an excellent environment to do more homologous recombination. We achieved HR frequencies that we never would have dreamed of. Being able to harness homologous recombination so easily now in plant cells was something that I didn’t think was going to be possible. So that was that was huge.    

Transforming leaf cells that the efficiency that we can transform them now in corn and other cereal crops was something that I thought would probably never happen. So, in my immediate area of work those are two impractical things early in my career that have become practical now. 

SS: Do you have anything which is impractical right now, would you like to see them to be practical in the future?

WGK: Two of the biggest areas, and there are a lot of people that are working hard on this right now are tissue culture free transformation and editing. Which kind of goes hand in hand in a lot of people’s minds now with DNA free editing techniques, using RNPs is the simplest example of that. But the development of nano technologies that are continuing to improve.

Some of the clever in plant transformation techniques that people are working on, some of the stuff that’s come out of the Voytas Lab is providing exciting first glimpses into what we might be able to do in that area. It’s just going to improve. You know tomorrow or the next day, a graduate student is going to wake up with some brilliant new idea.

SS: Yes. Tissue culture and somatic embryogenesis requires so much work to begin with that we need highly skilled people.  

WGK: Yes. You need to do that stuff. If you take maize transformation and editing as kind of an extreme example—just making the media for doing our experiments. For a graduate student, must come in and make up that media fresh every couple month, oh my gosh. If we could get around that, it would be tremendous. 

SS: My next question is about a recent report from the Department of Energy that titled—Overcoming Barriers in Plant Transformation, a focus on bioenergy crops. It was highlighted that lack of talent pool is a major obstacle for plant genetic engineering. Could you please reflect on any action plans that PlantGene is taking to fill that talent gap?

WGK: This is something that is across industry and academia. It is just become much more difficult to find people that are really interested and excited about this field. We are hoping that being able to provide this type of virtual interaction to a wider audience is going to hopefully continue to get more people excited.

We have a lot of work to do with universities that are working at the undergraduate level to try to get more undergrads excited about plant biotechnology and the opportunities. One of the big surprises for us, we had planned on having a series of virtual workshops but when we started providing these workshops, it became clear that it’s hard for people to find a lot of the basic information that they need to do this type of work. That’s why a lot of our virtual offering in the future is going to try to break this out into modules that we can provide to people going back to some of the basics. 

We have got one coming up here soon on handling agrobacterium. Sounds simple, but there are a lot of tricks and tips that you learn over the years. We want to make those available to people. We are hoping those types of things are going to be helpful.

We really want to go beyond just our steering committee and some of the colleagues that we are pulling in if we can get more volunteers like you who are willing to commit time and effort to helping with this virtual platform. Maybe it will continue to spread.

I’m hoping it will continue to spread, and we are hoping that it’s going to spread worldwide. Because through this kind of information exchange we can make experts in the community more accessible to students.

SS: I was reading that report that in the US, not that many centers where you can do your plant transformation. And even in our lab, like we want to do rice transformation. It’s so expensive we decided to do by ourselves right now.

WGK: Yeah, we need to keep working to make these methods more user friendly and more inexpensive. Simplify the methods as much as possible. As a first step we can continue to develop the WUSCHEL and BABY BOOM system. Focusing on leaf transformation to make it to make it accessible for a wide range of poaceae species.

Once we’ve done that, simplify the methods and simplify the media to the point where we could take these media formulations and give them to companies like PhytoTech. An undergrad would not have to come in and spend two days a month making media. They could just order powdered formulations from PhytoTech and make things up a lot more simply.

There are so many things that we need to improve. The transformation facilities around the country have hard to find and retain the high caliber of employees that they need to run these facilities. It’s becoming very prohibitive. Many of the transformation facilities instead of providing transformation services to anyone who wants them, which was the original model,

they’re having to fall back and basically just make their transformation services available to the university where they work.

SS: Which happens with Iowa State transformation facility—our lab used to send to Iowa State but now they are not doing. We send samples to another place.

WGK: Yeah. I hope that’s not going to be an increasing trend. We need to find some way to reverse that. We’re going to have to be creative.

SS: The last question is—This is an interesting and exciting time to be a plant scientist.

We, the PlantGene community and general audience would like to hear your advice to be a successful next generation plant scientist.

WGK: Well, be inquisitive, be persistent, and pay attention to the basics. I don’t think nowadays you can be restrictive in terms of the type of literature that you read. You must try to develop as broad spectrum of interest as possible. That goes beyond developmental biology. What is happening in the animal field that is analogous to things that we are interested in doing in plants.

What types of new material sciences are available or what types of new materials are coming out of engineering departments that are going to be useful for in vitro applications. How methods like 3D printing help us change our tissue culture methods. 

I think there is so many ways to be creative in this field. You just must keep your mind really open to possibilities.

SS: I see the cell programing is an emerging field. You already have done those things, but the name did not exist before. Also, synthetic biology. 

With that thank you for your time. I really appreciate it. I have more questions, but we can keep that for the next time.

WGK: Okay, well, I hope that this is going to be useful to people. It was fun. 

Footnote: PlantGene would like to thank the National Science Foundation for their support. PlantGene is funded through NSF Grant IOS2210962. If you would like to learn more about PlantGene or become a member, please visit www.plantgene.sivp.org