Phytofactories = Plant Factories
Phytofactories 2023, organized by the Luxembourg Institute for Science and Technology (LIST) took place in Luxembourg on 7-9th of June. Phytofactories 2023 was a two- and half-day focused conference about the science and technologies potentiating the development of known and novel high value products from plants. From small and lush mosses (Bryophytes) to trees, plants are, as we wrote before in one of our blogs, “natures’ alchemists” and we are just the apprentices, learning about the rich and still unexploited diversity of bioactive metabolites from plants. This blog was inspired by the Phytofactories conference.
As we know, the way we have grown plants for human use, as monocultures occupying extensive areas, is unsustainable. We need to develop alternative means of producing plants and trees (or the products that they afford us) that the growing human population needs, introducing technologies that do not cause more harm to the environment – and preferably, with technologies that mitigate the trends exacerbating the climate crisis. New agricultural practices that are more sustainable and equally or more efficient than current using biorefinery and circular strategies of production are being developed and are expected to improve sustainability of our crop production systems. The new technologies need to enable people to make food, feed and all the products and materials needed, without increasing the surface of cultivated land.
Plant cell cultures have been used for many years now by scientists, as biological models for studying important mechanisms of plant development and the complex and valuable products of plant secondary metabolism. Plant cell cultures have also been used as source material for production of cancer pharmaceuticals (paclitaxel) and bioactives used in cosmetics, and they are being explored for production of many more high value medicinal, nutrition and cosmetic products and in cellular agriculture, for food.
Phytofactories 2023 covered:
- the production of high value compounds from plant cell cultures, and use of biorefinery concept for production of high value products from currently un-exploited side streams in production crops,
- novel developments in cell culture techniques and processes, and cell culture as tools and models to study important plant cell processes,
- Germplasm and biodiversity conservation by cryopreservation
- Plant genome editing and metabolic pathway engineering for production of high value compounds in plants and plant cell cultures.
Multiple commercial applications for plant cell cultures
The knowledge about the natural pathways of plant secondary metabolism has been used to guide which plant organs (leaves, seeds, fruits, etc.) are used to extract the compounds of interest. Collecting plants grown outdoors has been the traditional way of sourcing these high value products, but the processes developed are limited by the growth conditions of the crop and by the amounts naturally accumulated in these tissues. Often, side streams from these processes are still rich in valuable compounds, so a biorefinery approach can be used. An alternative approach to using field plant materials, is to establish plant cell cultures, that can be grown as sources of valuable products and even as food, as their nutrition profile can be very interesting.
As we recently wrote, cellular agriculture, i.e., the development of processes whereby cells and not whole organisms constitute the product is one such alternative technology being developed. Cellular agriculture can address, for example, sourcing plants that are being threatened by the climate change impact on their natural habitats.
The Finnish company Fazer is using cacao cell cultures as a food ingredient, to mitigate the troubles with the current cocoa supply chains. The US-based company California Cultured raised 4M USD in 2021 to develop cacao plant cell-based products. And in Zurich, Switzerland, ZHAW’s “Center of cocoa and chocolate competences” is using cacao cell cultures to make chocolate that tastes like…good chocolate.
Coffee cell cultures and various berry cell cultures have been established at the VTT in Finland, with the aim of being used as food. A biorefinery approach in the case of plant cell cultures can be foreseen, in particular if the cells secrete compounds of high value to the culture media. Plant cell cultures may also be manipulated to elicit production of such compounds. Examples of these types of approach discussed during Phytofactories 2023 included development of apple cell cultures to be used as a source of antimicrobial compounds, and the establishment of cloud berry cell cultures and chicory hairy root cultures also as potential sources for antimicrobials. Compounds with important activity such as anti MRSA (methicillin-resistant Staphylococcus aureus) antimicrobial activity have been identified in these cell cultures.
Scaling up Plant cell cultures
Many proof of concept examples for using plant cell cultures as sources of food and of valuable compounds exist at laboratory scale (<1L to 2L culture volumes), but there are remarkably fewer examples of scaling-up these productions. Scaling up plant cell cultures presents specific challenges, related for example to the fact that the conditions for cell culture establishment and growth need to be established for each species, that there is a need for relatively large innocula for the large-scale cultures and that genetic instability has been observed in long-term cultures. Therefore, current practices tend to favour parallelization of smaller scale production runs and frequent start of cultures, from preserved calli or cell culture stocks.
Currently, paclitaxel (a cancer medicine) production by the company Phyton in steel bioreactors at 75000L scale is the largest commercial process scale for plant cell cultures. The VTT has scaled its coffee cell production to >1000L scale, which is much larger than many other processes, though it is understood that the food applications will require one or two orders of magnitude higher production volumes when at commercial scale. Other plant cell culture processes that have been scaled to 300L-400L include the approved Protalix recombinant therapeutic product (taliglucerase alfa) for enzyme replacement therapy in patients with Type I Gaucher disease.
These cell cultures are done in large flexible polyethylene bags, to which growth medium and air were supplied from a central system under sterile conditions (the ProCellEx™ system) at 400L scale. In this system such bioreactors can be serially assembled within a clean-room facility, allowing the growth of thousands of litres of transformed plant cells expressing the required recombinant protein at industrial scale.
The apple cell cultures producing antimicrobials at LIST have been scaled to 300L.
The cosmetics industry has been an earlier adopter of plant cell cultures as ingredients in their products. Mitsui Chemical was a pioneer in the 1980s with its 750L stirred air-life bioreactor production of shikonin from Lithospermum erythrorhizon Siebold & Zucc cell suspension culture. Shikonin is a red dye used in cosmetics as well as in food and for dyeing fabrics, and used in traditional Chinese medicine. Shikonin has anti-inflammatory and anti-cancer effect, though the compound is not approved as an active pharmaceutical ingredient. The status of commercially availability of shikonin is not currently clear. The company Mibelle Biochemistry developed the technology platform PhytoCellTec™ which “enables the large-scale cultivation of callus (stem) cells from rare and protected plant species”. These plant cell cultures are grown in modified 50 and 100 L disposable wave bioreactors.
The company ABR in Italy has also commercialized compounds from plant-cell cultures: the bioactives Teupolioside, Verbascoside, Echinacoside produced using ABR’s platform NATRISE ® which affords 2000kg of cell biomass and 80kg of botanical active per day. Several other companies globally are using plant cell cultures to produce valuable cosmetic products. Ginseng is traditionally used in Chinese and other traditional medicine practices. Nitto Denko Corporation scaled up the cultivation of Panax ginseng cell cultures to a 2000L scale, achieving 19 g/L dry cell biomass in merely 4 weeks, and receiving permission for commercial use of the manufactured biomass as a food additive in Japan in the late 1980s. Panax ginseng adventitious root culture was done in 1000 L balloon type air-lift bioreactor.
Commercially available bioreactors have not been developed with plant cell cultures in mind, but plant cell cultures could be relatively easily adapted to grow in both steel bioreactors and to the more recent single use bioreactors. As mentioned, the current largest scale for commercial plant cell culture is 75000L steel culture for paclitaxel. The 2000L disposable bags available for commercial disposable bioreactors have not yet been tested for plant cell cultures. On the other hand, there has been extensive use of single use bioreactors with bags of various types at scales up to 400L.
As the use of plant-cell cultures becomes more widespread in new applications, we will likely be seeing innovations in “plant cell-purposed” adaptations of the bioreactors.
Germplasm refers to the genetic information kept in safe and accessible banks that are thus “collections of genetic resources”. The US National Institute of Health further defines plants, seed, or cultures as “germplasm when maintained for the purposes of studying, managing, or using the genetic information they possess.”
Maintenance of germplasm is an important means of preserving biodiversity.
“There are approximately half a million plants on earth and of these around 50,000 species are used globally for food, feed, fiber, medicine and horticulture. It is estimated that at least 21% of all known vascular plants are either threatened, endangered or at the risk of extinction due to habitat loss, overexploitation, and the rapidly changing climate.”Dr. Praveen K. Saxena, Gosling Research Institute for Plant Preservation (GRIPP), University of Guelph, Canada
While seed banks are a useful means of preserving germplasm, seeds may not be available for all plant species, as some seeds are viable only for a short time and cannot be preserved. For some plant species we do not even have access to the seeds.
In vitro technologies can play an important role in preserving biodiversity and enhancing plant populations in natural habitats. Micropropagation is an advanced plant tissue culture technique, that is used to maintain living germplasm and produce large quantities of plants for replenishment, conservation and global distribution of endangered species and crops of economic importance. Cryopreservation allows the storage of genetic material at an ultra-low temperature (-160 °C or −196 °C), and the tissues can be maintained for decades with minimal loss of viability and genetic uniformity. Over 200 plant species including staple food crops, endangered species and plants of horticultural importance have been cryopreserved with varying degree of success. Integrated plant systems utilizing micropropagation and cryopreservation technologies hold great potential in mitigating the impact of current ecological crisis while complementing global conservation strategies.
Micropropagation is a valuable business with a global value estimated in the order of 1.5 Billion USD. Micropropagation allows for plant material free of diseases to be propagated and distributed. At scale, micropropagation makes use of specific techniques involving TIS – temporary immersion systems. These purpose-built bioreactors provide micropropagation techniques with the possibility of temporarily immersing the plant materials and small in vitro shoots in media, which is necessary because the alternative of keeping them immersed constantly as in the case of plant-cell cultures, is not viable in these cases.
Several TIS bioreactors have been developed. PlantForm in Sweden, Setis-systems in Belgium, Plant Cells Technologies in the USA, Vitropic and Cid-Plastiques in France are all companies commercializing various modalities of TIS bioreactors.
Cryopreservation enables preservation of many plant tissues and organs, including plant shoot meristems. When needed, the shoot meristems are brought back to culure temperatures and can be used in micropropagation and in breeding programs. Cryopreservation of calli and established cell cultures is important, as a source of “original” and disease-free material for plant culture. More than 80% of the existing cryopreserved accessions belong to five crops: potato, cassava, bananas, mulberry, and garlic. Other important plant cryopreservation collections representing thousands of accessions are those of dormant apple buds. Cryopreservation techniques are now used for plant germplasm storage in many institutes around the world. Several cryopreservation germplasm repositories (cryobanks) have been established for various plant species in different countries (e.g., cassava, potato, banana, apple, pear, coffee, mulberry, garlic), applying different cryopreservation techniques, and this strategy represents a guide for conservation in the future. The CGIAR Centers – notably Alliance Biodiversity International and CIAT, CIP and IITA – have cryopreserved collections. Other genebanks and institutions are also engaged in cryobanking in countries from EU, Japan and USDA. Bioversity International and KU Leuven with support of the Crop Trust, developed cryopreservation protocols for >30 species.
The CGIAR conducted in 2017, with various international centers a Feasibility Study for a safety backup for a cryopreservation facility.
The Feasibility Study issued the following recommendations:
– a major global initiative should be launched to accelerate the development and implementation of crop cryopreservation for important crops (estimate that 100 000 accessions need to be cryopreserved)
– a backup cryopreservation facility should be set up to accommodate the estimated 10 000 accessions that are already cryopreserved (cfr Svalbard Seed Vault).