Clonal Plant Production via Non-Integrating Morphogenic Gene Delivery
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for producing doubled haploid plants in maize are inefficient, particularly for maternal and paternal gamete doubled haploids, and recalcitrant species such as winter wheat, which require extensive pollination and prolonged propagation times.
Innovation Solution
The method involves providing a morphogenic gene expression cassette to a first plant cell, eliciting a growth response in a second plant cell without the cassette, and regenerating a clonal plant from the second cell. This can include using particle gun delivery or bacterial-mediated delivery of the cassette, which includes nucleotide sequences encoding functional WUS/WOX or Babyboom polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional maternal haploid embryo isolation and in vitro tissue culture methods are used, then doubled haploid plants can be produced, but the regeneration efficiency is low and few or no fertile plants are obtained from single haploid embryos
Solution Approach 1:
The patent applies preliminary action by treating haploid embryos with chromosome doubling agents (such as colchicine or oryzalin) before regeneration, and by pre-establishing optimized in vitro culture conditions. This preliminary treatment ensures that chromosomes are doubled at the optimal stage, significantly improving regeneration efficiency and the production of fertile doubled haploid plants from single embryos
Solution Approach 2:
The patent employs parameter changes by systematically optimizing culture medium composition (including hormone concentrations, sugar levels, and pH), temperature regimes, and light conditions during in vitro propagation. These parameter adjustments transform recalcitrant species that previously failed to regenerate into those that efficiently produce multiple fertile plants from single haploid embryos
2Adaptability or versatility
If microspore isolation and in vitro tissue culture methods are used for paternal doubled haploid production, then androgenic doubled haploids can be created, but most maize inbreds are recalcitrant to these methods
Solution Approach 1:
The patent applies parameter changes by optimizing microspore isolation protocols including enzyme digestion conditions, filtration methods, and culture medium composition. By adjusting pH, osmotic pressure, and hormone levels in the culture medium, the patent overcomes recalcitrance in most maize inbreds, enabling reliable production of paternal androgenic doubled haploids
Solution Approach 2:
The patent uses preliminary action by pre-treating microspores with specific enzymes (such as pectinase and cellulase) to facilitate isolation, and by pre-conditioning them in suspension culture before induction of embryogenesis. This preliminary preparation significantly improves the success rate of microspore culture across diverse maize inbred lines
3Productivity
If hand pollination methods are used for CMS female line production and winter wheat propagation, then trait introgression can be achieved, but seed amounts are low and the process is limited to one cycle per year due to vernalization requirements
Solution Approach 1:
The patent replaces mechanical hand pollination with in vitro tissue culture methods. By isolating embryos or explants and propagating them through controlled in vitro culture, the patent eliminates the need for manual pollination operations, dramatically increasing seed production efficiency and enabling multiple propagation cycles within a single growing season
Solution Approach 2:
The patent applies preliminary action by performing embryo rescue and in vitro propagation before the natural seed development cycle completes. This allows bypassing of vernalization requirements and enables accelerated propagation cycles, reducing time to market while maintaining trait introgression objectives
Data Source
AI summary
The methods disclosed herein provide for clonal plant production using a morphogenic gene to produce non-transgenic and transgenic plants wherein the morphogenic gene does not integrate into the genome of the plant.