Agrobacterium-mediated and particle bombardment transformation method for cowpea and dry bean meristem explants
AssigneeWisconsin Alumni Research Foundation
InventorsMichael William Petersen, Brian Joseph Martinell, Edward James Williams, Shawn Michael Kaeppler, Heidi F. Kaeppler
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Abstract
The present disclosure provides methods for the stable transformation of meristem explants from cowpeas (Vigna unguiculata) and dry beans (Phaseolus vulgaris).
Claims21
A method of transforming a cowpea, of the genus Vigna, or dry bean seed, of the genus Phaseolus, the method comprising the steps of
- rehydrating a dry cowpea or dry bean seed in a hydration medium for at least 6 hours to generate a rehydrated seed having a moisture content of between 30% and 75%,
- excising meristematic tissue from the rehydrated seed directly after rehydration to form an explant, wherein the excision removes the seed coat and cotyledons, and
- transforming the incubated explant with a heterologous nucleic acid of interest.
A method of transforming a cowpea, of the genus Vigna, or dry bean seed, of the genus Phaseolus, the method comprising the steps of,
- surface sterilizing a dry cowpea or dry bean seed,
- rehydrating the dry cowpea or dry bean seed in a hydration medium for at least 6 hours to generate a rehydrated seed having a moisture content of between 30% and 75%,
- excising meristematic tissue from the rehydrated seed directly after rehydration to form an explant, wherein the excision removes the seed coat and cotyledons,
- incubating the explant in an incubation medium prior to the transforming step, wherein the incubation medium is sterile and comprises 20% PEG4000 with 60 mg/L Captan fungicide and 30 mg/L Chlorothalonil fungicide, and
- transforming the explant with a heterologous nucleic acid of interest.
A method of transforming a cowpea, of the genus Vigna, or dry bean seed, of the genus Phaseolus, the method comprising the steps of,
- surface sterilizing a dry cowpea or dry bean seed,
- rehydrating the dry cowpea or dry bean seed in a hydration medium for at least 6 hours to generate a rehydrated seed having a moisture content of between 30% and 75%,
- re-drying the hydrated cowpea or dry bean seed for at least 2 hours to a moisture content less than 25%,
- excising meristematic tissue from the re-dried seed to form an explant wherein the excision removes the seed coat and cotyledons, and
- transforming the explant with a heterologous nucleic acid of interest.
A method of transforming a cowpea, of the genus Vigna, or dry bean seed, of the genus Phaseolus, the method comprising the steps of
- surface sterilizing a dry cowpea or dry bean seed in a liquid for at least 6 hours to generate a surface sterilized seed having a moisture content of between 30% and 75%,
- re-drying the dry cowpea or dry bean seed for at least 2 hours to a moisture content less than 25%,
- excising meristematic tissue from the rehydrated seed to form an explant, wherein the excision removes the seed coat and cotyledons and
- transforming the incubated explant with a heterologous nucleic acid of interest.
Dependencies are inferred from the claim text.