Monocot leaf explant preparation

Patent metadata

Field	Value
Publication number	US-2025134021-A1
Application number	US-202318835293-A
Country	US
Kind code	A1
Filing date	Feb 3, 2023
Priority date	Feb 3, 2022
Publication date	May 1, 2025
Grant date	—

Abstract

Methods, including high-throughput, automated methods for preparing, by mechanical and/or robotic means, monocot leaf explants for transformation, are provided.

First claim

That which is claimed:

1 . A high-throughput, automated method of producing a population of leaf explants comprising:
sterilizing a population of seed to provide a sterilized seed population; transferring the sterilized seed population by mechanical and/or robotic means to a germination container, wherein the germination container comprises a germination medium; providing a distribution of the sterilized seed population by mechanical and/or robotic means in the germination container; germinating the sterilized seed population to provide a population of germinated seedlings; growing the germinated seedlings to a sufficient height for harvesting seedling leaves by mechanical and/or robotic means; harvesting the seedling leaves by mechanical and/or robotic means to provide harvested seedling leaves; transferring the harvested seedling leaves to a holding container by mechanical and/or robotic means, wherein the holding container comprises a chopping means, a cutting means, and/or a blending means and chopping, cutting, and/or blending the harvested seedling leaves to provide leaf segments of an appropriate size for a bacterial infection; providing to the holding container a bacterial infection solution; infecting the leaf segments in the holding container for a sufficient amount of time to provide bacterially infected leaf segments; removing by mechanical and/or robotic means the bacterial solution from the bacterially infected leaf segments; and providing further sub-culturing, resting, selection, heat-treatment, regeneration, maturation, or rooting processes by automated, mechanical, and/or robotic means.

2 . The method of claim 1 , wherein the sterilizing comprises an ethanol treatment, a chlorine gas treatment, a dilute bleach solution treatment, and combinations of the foregoing.

3 . The method of claim 1 , wherein the germination medium is a solid, a semi-solid, or a hydroponic medium.

4 . The method of claim 1 , wherein harvesting comprises cutting and/or chopping the harvested seedling leaves with scissors, blades, wires, lasers, high-pressure water jet streams, high-pressure air, and combinations of the foregoing.

5 . The method of claim 1 , wherein the holding container further comprises a bacterial infection solution.

6 . The method of claim 5 , wherein the bacterial infection solution comprises Agrobacterium.

7 . The method of claim 1 , wherein the chopping, cutting and or blending of the seedling leaves is performed in a blender or a food processor.

8 . The method of claim 1 , wherein the leaf segments of an appropriate size comprise leaf segments of from about 0.1 mm to about 1 mm, from about 1 mm to about 2 mm, from about 2 mm to about 3 mm, or from about 3 mm to about 1 cm in size.

9 . A high-throughput, automated method of producing a population of leaf explants comprising:
transporting each seedling from a population of seedlings to a first cutting device; removing a top portion of the seedlings using the first cutting device; transporting the seedlings to a second cutting device after removing the top portion of the seedlings; removing a second portion of the seedlings using the second cutting device to provide leaf segments; loading the leaf segments into a loading device configured to dispense a predetermined amount of leaf segments into a container each comprising means for chopping, a cutting, and/or a blending the leaf segments; dispensing the predetermined amount of leaf segments in the container; and chopping, a cutting, and/or a blending the leaf segments to provide leaf fragments of a predetermined size.

10 . The method of claim 9 , wherein the transporting steps includes using a conveyor.

11 . The method of claim 10 , further comprising
measuring a height of each seedling included in the population of seedlings using an optical sensor; determining if the measured height of the seedlings is within a predetermined height threshold; and transporting each seedling to the first cutting device if the measured height of the seedlings is within the predetermined height threshold.

12 . The method of claim 11 , further comprising
measuring a length of each leaf segment using an optical sensor after removing the second portion of the seedlings; determining if the measured length of each leaf segment is within a predetermined length threshold; and loading the leaf segments into the loading device if the measured length of the leaf segment is within the predetermined length threshold.

13 . The method of claim 9 , wherein the first cutting device includes one of scissors, blades, wires, lasers, high-pressure water jet streams, and high-pressure air.

14 . The method of claim 9 , wherein the second cutting device includes one of scissors, blades, wires, lasers, high-pressure water jet streams, and high-pressure air.

15 . The method of claim 9 , further comprising:
injecting a predetermined amount of bacterial infection solution into the container and attaching a cap to the container to close the container and prevent the leaf segments and bacterial infection solution from moving outside the container.

Assignees

• Pioneer Hi Bred Int

Inventors

• Gordon-Kamm William James
• Hayes Thomas Richard
• Maurer Brandon
• Ryan Larisa A
• Sardesai Nagesh
• Wang Ning
• Wu Xinli Emily

Classifications

• C12N15/8205

  Agrobacterium mediated transformation · CPC title

• A01G22/00

  Cultivation of specific crops or plants not otherwise provided for · CPC title

• A01C1/02

  Germinating apparatus; Determining germination capacity of seeds or the like · CPC title

• A01H4/008 Primary

  Methods for regeneration to complete plants · CPC title

• A01H4/00 Primary

  Plant reproduction by tissue culture techniques {; Tissue culture techniques therefor} · CPC title