Team:HebrewU/Open Source
Hebrew- Project
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We created an open source platform aimed at helping researchers and organizations worldwide tackle their dioxin pollution. In our open source page, we present the basic foundations necessary to create a viable transgenic plant based on our research and experiments.
Our open source page features two main programs: our synthetic enzymatic pathway (i.e. optimized gene sequences for each plant, including descriptions), and our proposed plants that are optimal for our pathway.
We chose the plants based on 4 categories: Transformation, dioxin uptake efficiency, sterilization methods, and plant durability. We did our best to gather the necessary information to support it using both academic research and verified gardening sites.
Transformation - How many methods are known for the transformation of this plant? How easy and effective are they?
Dioxin uptake efficiency - How well does the plant absorb dioxin from the ground, compared to other plants?
Sterilization methods - With these types of solutions, it is important to be ecologically responsible; releasing genetically engineered plants into the environment is no trivial matter. The ability to sterilize the plants, along with short life expectancies in general, is key for the safe release of transformed plants.
Plant durability - Under what conditions do the plants grow? Where do they grow? Do they grow in regions that are in dire need of solutions to dioxin contaminations?
Cucurbita pepo L. (Pumpkin & Zucchini)
- Deep root systems.
- Zucchini roots grow mainly downward, while pumpkin`s grow mainly outward.
- High uptake values of nutrition from soil.
- Able to absorb dioxins through roots and deliver them throughout the plant (potential for high-speed degradation in the plant).
- Short-season plants, grown in hot seasons.
- Easily grown in most areas of the world.
- Existing transformation methods use agrobacterium.
- Whole-genome sequence doesn`t exist yet.
- No specific method was found.
1 Uptake Rate
As shown in the figure below, zucchini has shown an impressive absorbance ability of dioxins from contaminated soil11. The main advantage zucchini and pumpkin plants have over other plants (that have been studied) are their ability to transfer the dioxins to their shoots, flowers, and fruits, while others cannot2, so theoretically we have more dioxin to mass ratio, letting them absorb more dioxins. As such, zucchini and pumpkin are our main candidates for dioxin phytoremediation in case of a suitable climate.
"Plants have been frequently shown to remove POPs from soils (Zhao et al. 2006; Susarla et al. 2002; Macek et al. 2000). The high propensity of selected Cucurbitaceae to extract PCDDs/PCDFs from soil was first reported by Hülster et al. (1994), who found that C. pepo L. fruits contained double the PCDD/PCDF concentrations of other examined plants."1
Fig. 1:
"Mean decreases in total and TEQ PCDD/PCDF concentrations in soil amended with different doses of sewage sludge (A1, A2) and urban sediments (B1–B2) before and after Cucurbita pepo L. cv ‘Atena Polka’ cultivation".3
2 Growth Conditions
Zucchini and pumpkin are very sensitive to cold weather, requires warm temperatures of above 16 degrees Celsius to grow, but to warm of a climate (above 38 degrees) will harm the plant. Zucchini takes about 60 days to mature. By then it produces fruit as long as someone picks them before they rot. Pumpkin requires between 90 to 160 days to mature. They both grow relatively quickly and, as such, have high root-uptake of nutrition from the ground. Both require moderate quantities of water to grow, which may make it more difficult to grow in drier climates; however, pumpkins have been successfully grown in Iraq, for example. Zucchini roots generally extend deeply downward in a taproot formation, while pumpkin roots tend to spread horizontally as well as vertically - most of its nutrients are absorbed from the upper 1/2 meter of the soil. 3,4,5
3 Plant Transformation:
We know of a few agrobacterium strains able to transform pumpkin plants and zucchini6,7. Agrobacterium is one of the most popular methods of plants transformation, widely studied in plant research.
4 Plant Sterility
Triploid plants* have larger organs, greater biomass, and strong stress resistance by preserving relatively larger amounts of photosynthetic energy8. The undesirable spread of non-native invasive crop and horticultural plants into natural areas can also be reduced or eliminated by the use of triploids because they tend to be sterile and seedless There are few different ways to create triploid plants:
- - Natural selection - triploid plants occur in nature, meaning it is possible to look for the desired plant in nature.
- - Artificial hybridization - by sexual hybridization of different ploidy, one can create a triploid offspring.
- - Endosperm culture in vitro - endosperm is a triploid tissue. Successful regeneration of a plant from an endosperm tissue would result in a triploid plant.
• Triploid plants contain 3 sets of chromosomes, meaning they have 3n chromosomes. Triploid organisms, in general, tend to be sterile, as in most plants.
Optimized genes for Cucurbita pepo:
* In order to optimize the genes, we used our MOOLTi codon optimizer. We originally designed MOOLTi to perform optimizations for multiple organisms simultaneously, but it is also capable of optimizing for a single organism with ease.
References:
1. "Potential for Phytoremediation of PCDD/PCDF-Contaminated Sludge and Sediments Using Cucurbitaceae Plants: A Pilot Study" Magdalena Urbaniak et al. (2016).
2. "Uptake by roots and translocation to shoots of polychlorinated dibenzo-p-dioxins and dibenzofurans in typical crop plants" Haijun Zhang et al. (2009).
3. "Planting, growing, and harvesting pumpking" The Old Farmer'S Almanac website (2010).
4. "How to Grow Zucchini: A Squash Vocabulary Lesson"
Charity Shumway (2012).
5. "What Is the Life Expectancy of Zucchini Plants?" Karen Carter.
6. "Cucurbita pepo L. can be transformed by Agrobacterium rhizogenes" Luigi Sanità di Toppi et al. (1997).
7. "A simple method for transient transformation of pumpkin (Cucurbita maxima) seedlings" Francisco Arturo Ramírez-Ortega et al. (2015).
8. "Breeding Triploid Plants: A Review"
Xiling Wang et al. (2016).
Brassica rapa (Chinese cabbage)
- Roots do not extend deeply or widely relative to other plants.
- High uptake of dioxins in roots.
- Very low transfer of dioxins from roots to shoots.
- Grows in most parts of the world, mostly in temperate-cold areas.
- Grown all year round.
- An abundance of agrobacterium strains known to be effective.
- Whole genome sequenced1.
- Susceptible to hybridization.
- Known male and female sterile mutants.
1 Uptake Rate
Although zucchini and pumpkin have shown the best overall uptake of dioxins, primarily showing an extraordinary shoot uptake of dioxins, Chinese cabbage has shown an especially high uptake of dioxins in their roots only according to Table 22. Since our proposed method takes effect in roots as well as the shoots, a high enough root uptake should suffice.
2 Growth Conditions
Chinese cabbage requires cool temperatures, between 7-17 degrees Celsius, to grow and it takes between 50-85 days to mature. It`s roots grow in taproot formation, spreading into about 30 cm radius and 75 cm deep. The deeper the roots are, the less branched they are. This plant doesn`t require a large amount of water and is able to keep growing without too much intervention.3
3 Plant Transformation:
An abundance of research exists that describes the transformation of Chinese cabbage using different agrobacterium strains, presenting 1-3% transformation rates. 4,5,6
4 Plant Sterility
Both female and male sterile strains exist for the Chinese cabbage7,8,9. The protocols for molecular techniques and the creation of hybrid strains are available for this plant.
"..…compared to the wild-type line ‘FT,’ the fsm plants exhibited pistil abortion. Whether the fsm mutant was self-pollinated or used as the female parent to accept foreign pollen (wild-type line ‘FT’), the seed setting rates of fsm were both zero. The results showed that the female sterility of fsm was stable."
Optimized genes for Brassica rapa:
* In order to optimize the genes, we used our MOOLTi codon optimizer. We originally designed MOOLTi to perform optimizations for multiple organisms simultaneously, but it is also capable of optimizing for a single organism with ease.
References:
1. "The genome of the mesopolyploid crop species Brassica rapa" Wang X and Zhang Z (Brassica rapa Genome Sequencing Project Consortium, 2011).
2. "Uptake by roots and translocation to shoots of polychlorinated dibenzo-p-dioxins and dibenzofurans in typical crop plants" Haijun Zhang et al. (2009).
3. "HOW TO GROW CHINESE CABBAGE" Steve Albert (2018).
4. "Agrobacterium-mediated transformation of cotyledonary explants of Chinese cabbage (Brassica campestris L. ssp. pekinensis)" F.-L, Zhang and M. Watanabe (2000).
5. "Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker" Min BW and Harn CH (2007).
6. "Agrobacterium-mediated transformation and regeneration of fertile transgenic plants of chinese cabbage (brassica campestris ssp. pekinensis cv. 'spring flavor')" Jun S 2nd and Paek KH(1995).
7. "Interspecific hybridisation of cytoplasmic male-sterile rapeseed with Oguracytoplasm and Brassica rapa var. pekinensis as a method to obtain male-sterile Chinese cabbage inbred lines" Piotr Kamiński et al. (2016).
8. "Transcriptome Analysis of a Female-sterile Mutant (fsm) in Chinese Cabbage (Brassica campestris ssp. pekinensis)" Shengnan Huang et al. (2017).
9. "SSR and SCAR mapping of a multiple-allele male-sterile gene in Chinese cabbage (Brassica rapa L.)" Feng and Lim YP (2009).
Solanum lycopersicum (Tomato)
- Roots grow deep and wide.
- Very low transfer of dioxins from roots to shoots.
- Grows in most parts of the world, requires direct sunlight.
- Grown all year round.
- Perennial plant.
- Very effective agrobacterium transformation (40%+ transformation frequency).
- Whole genome sequenced.1
- Known male and female sterile mutants.
- Known CRISPR methods for seedless fruits.
- Problem of vegetative growths regarding the spread of transgenic plants.
1 Uptake Rate
Much like the Chinese cabbage, tomato plants have high dioxin uptake in their roots but very poor transfer to shoots through the xylem2. The main difference between the two [plants] is higher root uptake in Chinese cabbage, while tomatoes have an easier transformation and longer life expectancy.
2 Growth Conditions
Tomato plants come in different sizes and varieties. Tomato plants are perennials, mostly requiring direct sunlight to grow and love warm temperatures, able to grow in most areas of the world. It`s roots form a taproot formation, reaching half a meter to a meter and a half deep and 1 meter in diameter.
3 Plant Transformation:
Plant transformation using agrobacterium is considered robust, with transformation frequencies of 40%+3. On top of that, a whole genome sequence, being continuously studied for several decades now, gives us superb knowledge of tomato transformation possibilities.
4 Plant Sterility
A technique for promoting fruit formation without seed creation using CRISPR/Cas9 exists4 as well as known male and female sterile strains5. On the downside, the plant is known for its ability to form roots everywhere on its stem, meaning it can easily spread by vegetative means.6
Optimized genes for Solanum lycopersicum:
* In order to optimize the genes, we used our MOOLTi codon optimizer. We originally designed MOOLTi to perform optimizations for multiple organisms simultaneously, but it is also capable of optimizing for a single organism with ease.
References:
1. "Tomato gene sequence" Fernandez-Pozo et al (2014).
2. "Uptake by roots and translocation to shoots of polychlorinated dibenzo-p-dioxins and dibenzofurans in typical crop plants" Haijun Zhang et al. Chemosphere; Volume 76, Issue 6, Pages 740-746 (2009).
3. "A simple and efficient Agrobacterium-mediated procedure for transformation of tomato" Sharma MK and Sharma AK (2009).
4. "Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9" Risa Ueta et al. Scientific Reports; volume 7, Article number: 507 (2017).
5. " SEXUAL STERILITY is Essential for Both Male and Female Gametogenesis in Tomato." Hao S et al. Plant Cell Physiol 58(1):22–34 (2017).
6. "Planting, growing, and harvesting tomatoes" The Old Farmer's Almanac website (2018).