Since these technologies are unlikely to be mission critical during Artemis, their TRL can be increased and their risk factors studied through in-space evaluation. The Artemis missions also provide a test bed to evaluate the space-based evolution of microbes and alterations of seed stocks as a risk inherent to the biological component of the biomanufactory. This risk can be mitigated by incorporating backup seed and microbial freezer stocks to reset the system. However, ensuring that native and/or engineered traits remain robust over time is critical to avoid the resource penalties that are inherent to such a reset. Consequently, while optimal organisms and traits can be identified and engineered prior to a mission, testing their long-term performance on future NASA missions prior to inclusion in life support systems will help to assess whether engineered traits are robust to off-planet growth, whether microbial communities are stable across crop generations, and whether the in situ challenges that astronauts will face when attempting to reset the biomanufacturing system are surmountable. Quantifying these uncertainties during autonomous and crewed Artemis missions will inform trade off and optimization studies during the design of an enhanced life support system for Martian surface bio-operations.Crewed surface operations of ∼ 30 sols by four to six astronauts are projected to begin in 2031 ,growing tomatoes hydroponically with an additional mission similar in profile in 2033 . Given the short duration, a mission-critical biomanufactory as described herein is unlikely to be deployed.
However, these short-term, crewed missions RMA-S1, S2 provide opportunities to increase the TRL of biomanufactory elements for ∼ 500 sol surface missions RMA-L1 in ∼ 2040 and RMA-L2 in ∼ 2044. Building on the abiotic ISRU from early Artemis missions, we propose that RMA-S1 carry experimental systems for C-and-N-fixation processes such that a realized biomanufactory element can be properly scaled . Since RMA-S1, S2 will be crewed, regolith process testing becomes more feasible to be tested onsite on the surface of Mars, than during a complex sample return mission. Additionally, while relying on prepacked food for consumption, astronauts in RMA-S1 will be able to advance the TRL of platform combinations of agriculture hardware, crop cultivars, and operational procedures. An example is growing crops under various conditions to validate that a plant microbiome can provide a prolonged benefit in enclosed systems, and to determine resiliency in the event of pathogen invasion or a loss of microbiome function due to evolution. Additionally, the TRL for crop systems can be re-evaluated on account of partial gravity and/or microgravity. The RMA-S1 and RMA-S2 crews will be exposed for the first time to surface conditions after interplanetary travel, allowing for an initial assessment of health effects that can be contrasted to operations on the lunar surface , and that may be alleviated by potential biomanufactory pharmaceutical and functional food outputs . The RMA-S1 and RMA-S2 mission ISRU and FPS experiments will also provide insight into the input requirements for downstream biomanufactory processes. ISM technologies such as bioplastic synthesis and additive manufacture can be evaluated for sufficient TRL. Further, loop closure performance for several desired products can also be tested. This will help estimate the impact of waste stream characteristics changes on recycling .We have outlined the design and future deployment of a biomanufactory to support human surface operations during a 500 days manned Mars mission.
We extended previous stand-alone biological elements with space use potential into an integrated biomanufacturing system by bringing together the important systems of ISRU, synthesis, and recycling, to yield food, pharmaceuticals, and bio-materials. We also provided an envelope of future design, testing, and biomanufactory element deployment in a roadmap that spans Earth-based system development, testing on the ISS, integration with lunar missions, and initial construction during shorter-term initial human forays on Mars. The innovations necessary to meet the challenges of low-cost, energy and mass efficient, closed-loop, and regenerable biomanufacturing for space will undoubtedly yield important contributions to forwarding sustainable biomanufacturing on Earth. We anticipate that the path towards instantiating a biomanufactory will be replete with science, engineering, and ethical challenges. But that is the excitement—part-and-parcel—of the journey to Mars.Doubled haploid techniques have established themselves as useful tools in plant breeding and genetic research. As of 2005 doubled haploids had been produced in over 230 different crops . DH lines can be produced in several ways, by distant hybridization , by androgenesis or by gynogenesis . Each method has some advantages and disadvantages and not all are applicable or efficient in all crops. As discussed by Forster and Thomas 2005 the advantage of DH in breeding is in time savings between the initial cross and large-scale testing of developed lines.The advantage of DHs in genetic research is that perfectly homozygous lines can be produced relatively quickly and permit large scale replicated trials for detection of quantitative trait loci and their allocation to specific, sometimes very small, chromosome regions. In all cases, the critical issue in the use of double haploids is the efficiency of their production. This efficiency can be measured in several ways but it can always be brought down to the ultimate factor: how many useful lines can be produced in a given time with available labor and resources. For those involved in the DH development, the critical measure of efficiency is the number of green plants produced.
However, not all green plants need to be useful in breeding or research. Given that stability/uniformity is the main advantage of the DH lines, any deviation from it, including that caused by aneuploidy, is detrimental and in most cases will disqualify a line from any research or breeding. Triticale is a new cereal that in ca. 135 years since the first man-made wheat-rye hybrids were created has established itself as a competitive crop in certain environments. Unlike its progenitors, wheat and rye, triticale is generally amenable to anther culture and in most instances produces reasonable yields of haploids. First commercial cultivars of triticale developed with the aid of androgenesis have been released and the method is routinely used in breeding with thousands of DHs produced every year . The androgenic response in triticale appears to be under genetic control but in most cases, sufficiently large populations can be generated. However, despite a considerable progress in breeding for meiotic stability, triticale still has chromosome pairing issues and tends to show a fair proportion of aneuploids among sexually derived progenies, more so in hybrids than established lines . Perhaps for this reason,hydroponic growing supplies the issue of efficiency of the DH production and the quality of the recovered DH lines requires more attention in triticale than in most better established and more meiotically stable crops, such as wheat or barley. The wider context of this study was to generate sufficiently sized mapping populations of the DH triticale lines. Aneuploidy severely complicated this effort and appeared as an issue deserving to be analyzed with an additional effort. However, since from the start the emphasis was on the mapping populations and not the process of androgenesis itself, some data on population sizes and frequencies of specific events were never collected. Still, it appears that the issue of aneuploidy in triticale DH lines is important enough to be addressed.The measurements of the DNA contents were done on a population of spontaneously DHs of the Presto 9 Mungis combination. Ploidy levels of the regenerated plants were evaluated by fluorescence using a Partec II flflow cytometer . Samples were prepared according to Galbraith et al. with some modifications. Leaf fragments were chopped with a sharp razor blade in a Petri dish with 2 ml of the nuclei-isolation buffer Triton X-100 containing DAPI and the cell suspension was filtered through a 30-lm filter. The ploidy levels of regenerants were determined by comparing the G1 peaks of the analyzed samples to internal controls, here both parents, cv. Presto and Mungis. Plants with the peaks of the DNA contents distribution clearly separated from the controls were considered aneuploid.Root tips for karyotyping were collected either from seed germinated on wet filter paper in Petri dishes, or from plants grown in a hydroponic culture in an aerated full strength GroResearch Gromagnon 9-5-18 All-Purpose-Nutrient solution from American Hydroponics, Arcata, CA USA. For collections from germinating seedlings, seed were placed on wet filter paper in Petri dishes for 2–3 days at room temperature, refrigerated at ca.2℃ for 2–3 days, transferred to room temperature for 24 h and collected to ice water for ca. 27 h. Depending on the intended cytological protocol, roots were either fixed in 45% acetic acid overnight and squashed, or fixed in a Carnoy solution .
Roots from the hydroponic-grown plants were collected to ice water for ca. 27 h and fixed in the Carnoy solution. For the analyses of meiotic metaphase I chromosome pairing one anther from a spikelet of an ear was excised and live-squashed in a drop of 1% acetocarmine. If MI was present, the remaining two anthers were fixed in the Carnoy solution. All material in the Carnoy solution was fixed for 7 days at 37℃, stained in 1% acetocarmine for 2 h, re-fifixed and stored at -20℃ until used. All preparations of this type of material were made by squashing in a drop of 45% acetic acid using the two-coverslip technique. All C-banding on the 45% acetic acid- fixed root tips was according to Lukaszewski and Xu ; all C-bandning on the Carnoy-fixed material, whether root tips or anthers, was according to Giraldez et al. . In situ probing with labeled DNA was done according to the protocol of Dr. T. Endo, Kyoto University, Japan . Total genomic DNA of rye was labeled with digoxigenin and detected by the antidigoxigenin-fluorescein using standard kits and protocols from Roche Applied Science . The probe was mixed with blocking wheat DNA prepared according to MasoudiNejad et al. , usually in the probe to block ratio of ca. 1:100. Counter staining was with 1% propidium iodide. Sequential C-banding- in situ probing were done using the protocol provided by Dr. T. Endo, Kyoto University, Japan. For sequential C-banding- in situ probing, preparations were made in the same fashion as for standard in situ probing, they were C-banded, analyzed and photographed. The slides were then de-stained air dried, and probed using the Masoudi-Nejad et al. protocol. The cells photographed after C-banding were re-photographed after the in situ probing. All observations were made under a Zeiss Axioscope 20 equipped with epi-fluorescence, recorded with a SPOT RT Color digital camera , and processed using the SPOT Advanced and Adobe Photoshop CS software to improve contrast and resolution.The larger purpose of this study was to generate useful DH lines and no detailed records of the regeneration rates/ success were collected for every attempt or in every season. The total number of green androgenic plants transplanted to soil exceeded 3,500. Weak green plants that did not grow well in test tubes were not transplanted and no record exists of their numbers/proportions. Of the transplants, a certain proportion died before the colchicine treatment and the weakest of the survivors were not colchicine-treated. The estimated overall proportion of weak green plants rejected before colchicine treatment was about 8%, but in different cross combinations it ranged from ca. 2–3% toca. 70% in the group of late regenerants in Presto 9 NE422T. Mortality due to colchicine was low . Seed set per colchicine-treated plant ranged from a single seed to complete fertility. Complete fertility is probably indicative of spontaneous chromosome doubling in early microspore divisions rather than an effect of colchicine. In three cycles of androgenesis that included 644 colchicine-treated plants for which fairly detailed counts were made, 157 green plants were deemed to be aneuploid, based on their morphology at flowering. Most of these presumed aneuploids did not set seed. Those that did set seed and had their progeny karyotyped, all were aneuploid. Among plants that appeared normal in C0 and set seed, additional 16 were deemed aneuploid based on their morphology and seed set in C1, and all were found to be aneuploid upon karyotyping. Therefore, the average frequency of aneuploids among all regenerated green plants from all cross combinations must have exceeded 35%. In one of the most recalcitrant combinations, NE422T 9 Stan 1, among 72 plants that survived colchicine treatment, 50 were morphologically abnormal and were considered aneuploids.