Plant regeneration from protoplasts

Claim 2

An in vitro method for preconditioning cultivated tomato plants that will be used as sources of protoplast donating plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured protoplasts, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from cultivated tomato plants, until a shoot extends from said seed, (b) growing said shoot from step (a) in a nutrient medium, to which no exogenous plant hormones have been added, until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness, (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with "pre-enzyme treatment" (PET) solution, said PET solution being comprised essentially of an osmoticum to which exogenous plant hormones have been added, and (f) using said excised, tomato plant tissue from step (e) as a source of protoplast donating tomato plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured tomato protoplasts.

Claim 3

A method according to claim 1 wherein said cultivated tomato plants are Lycopersicon esculentum tomato plants.

Claim 4

A method according to claim 2 wherein said Lycopersicon esculentum tomato plants are Lycopersicon esculentum cultivars selected from the group comprised of Lycopersicon esculentum cv. Red Cherry, Lycopersicon esculentum cv. Cocktail Cherry, Lycopersicon esculentum cv. VFNT Cherry, Lycopersicon esculentum cv. VF36, Lycopersicon esculentum cv. Manapal, Lycopersicon esculentum cv. Floradade, Lycopersicon esculentum cv. UC82, Lycopersicon esculentum cv. Roma, Lycopersicon esculentum cv. Beefsteak, and Lycopersicon esculentum cv. San Marzano.

Claim 5

A method according to claim 1 wherein said protoplast donating cultivated tomato plant seed of step (a) is germinated at about 25° C.

Claim 6

A method according to claim 1 wherein said shoot of step (b) is grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark).

Claim 7

A method according to claim 1 wherein said tomato plantlet of step (c) is exposed to total darkness for about 48 hours.

Claim 8

A method according to claim 6 wherein said tomato plantlet is maintained at about 25° C. while being exposed to total darkness.

Claim 9

A method according to claim 1 wherein the excised tomato plant tissue of step (d) is excised tomato plant tissue selected from the group consisting of leaf tissue, stem tissue, cotyledon tissue and root tissue.

Claim 10

A method according to claim 1 wherein said pre-enzyme treatment (PET) solution of step (e) contains plant hormones.

Claim 11

A method according to claim 9 wherein said plant hormones are plant hormones that encourage cell growth and differentiation.

Claim 12

A method according to claim 9 wherein said plant hormones are plant hormones selected from the group consisting of auxins and cytokinins.

Claim 13

A method according to claim 1 wherein said excised tomato plant tissue in step (e) is treated with said "pre-enzyme treatment" (PET) solution for about 8-14 hours.

Claim 14

A method according to claim 1 wherein said "pre-enzyme treatment" (PET) solution of step (e) is chilled.

Claim 15

A method according to claim 13 wherein said chilled "pre-enzyme treatment" (PET) solution is chilled to about 10° C.

Claim 16

An improved method for regenerating cultivated tomato plants from cultured tomato protoplasts, said regeneration method including the steps of: (1) isolating tomato protoplasts from protoplast donating tomato plant tissue derived from cultivated tomato plants, (2) culturing said protoplasts from step (1), in growth media containing plant hormones that encourage cell growth, until said protoplasts divide and form small multi-cellular colonies composed of about 100 protoplast-derived cells growing in close proximity to one another, said small multi-cellular colonies not being visible to the naked eye, (3) culturing said small multi-cellular colonies from step (2), in growth media containing plant hormones that encourage cell growth, until said small multi-cellular colonies from small mini-calli that are about 3-5 mm in diameter, and visible to the naked eye, (4) culturing said small mini-calli from step (3), in differentiation media containing plant hormones that encourage differentiation and further growth, for about one week, (5) culturing the calli from step (4) in differentiation media, containing plant hormones that encourage further differentiation and growth of said calli, until shoots grow from said calli, (6) removing said shoots from said shoot-bearing differentiated calli of step (5), (7) culturing said shoots from step (6), in growth media containing plant hormones that encourage root formation, until said shoots develop roots, and (8) growing said rooted shoots from step (7) until said rooted shoots become tomato plants; wherein the improvement comprises an in vitro method for preconditioning a protoplast donating cultivated tomato plant prior to protoplast isolation, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from cultivated tomato plants, until a shoot extends from said seed, (b) growing said shoot from step (a) in a nutrient medium, to which no exogenous plant hormones have been added, until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, exposing said tomato plantlet from step (b) to total darkness, (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with "pre-enzyme treatment" (PET) solution, being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, and (f) using said excised, tomato plant tissue from step (e) as a source of protoplast donating tomato plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured tomato protoplasts.

Claim 17

An improved method for regenerating cultivated tomato plants from cultured tomato protoplasts, said regeneration method including the steps of: (1) isolating tomato protoplasts from protoplast donating tomato plant tissue derived from cultivated tomato plants, (2) culturing said protoplasts from step (1), in growth media containing plant hormones that encourage cell growth, until said protoplasts divide and form small multi-cellular colonies composed of about 100 protoplast-derived cells growing in close proximity to one another, said small multi-cellular colonies not being visible to the naked eye, (3) culturing said small multi-cellular colonies from step (2), in growth media containing plant hormones that encourage cell growth, until said small multi-cellular colonies from small mini-calli that are about 3-5 mm in diameter, and visible to the naked eye, (4) culturing said small mini-calli from step (3), in differentiation media containing plant hormones that encourage differentiation and further growth, for about one week, (5) culturing the calli from step (4) in differentiation media, containing plant hormones that encourage further differentiation and growth of said calli, until shoots grow from said calli, (6) removing said shoots from said shoot-bearing differentiated calli of step (5), (7) culturing said shoots from step (6), in growth media containing plant hormones that encourage root formation, until said shoots develop roots, and (8) growing said rooted shoots from step (7) until said rooted shoots become tomato plants; wherein the improvement comprises an in vitro method for preconditioning a protoplast donating cultivated tomato plant prior to protoplast isolation, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from cultivated tomato plants, until a shoot extends from said seed, (b) growing said shoot from step (a) in a nutrient medium, to which no exogenous plant hormones have been added, until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, exposing said tomato plantlet from step (b) to total darkness, (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with "pre-enzyme treatment" (PET) solution, being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, and (f) using said excised, tomato plant tissue from step (e) as a source of protoplast donating tomato plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured tomato protoplasts.

Claim 18

A method according to claim 15 wherein said cultivated tomato plants are Lycopersicon esculentum tomato plants.

Claim 19

A method according to claim 16 wherein said Lycopersicon esculentum tomato plants are Lycopersicon esculentum cultivars selected from the group comprised of Lycopersicon esculentum cv. Red Cherry, Lycopersicon esculentum cv. Cocktail Cherry, Lycopersicon esculentum cv. VFNT Cherry, Lycopersicon esculentum cv. VF36, Lycopersicon esculentum cv. Manapal, Lycopersicon esculentum cv. Floradade, Lycopersicon esculentum cv. UC82, Lycopersicon esculentum cv. Roma, Lycopersicon esculentum cv. Beefsteak, and Lycopersicon esculentum cv. San Marzano.

Claim 20

A method according to claim 15 wherein said protoplast donating cultivated tomato plant seed of step (a) is germinated at about 25° C.

Claim 21

A method according to claim 15 wherein said shoot of step (b) is grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark).

Claim 22

A method according to claim 15 wherein said tomato plantlet of step (c) is exposed to total darkness for about 48 hours.

Claim 23

A method according to claim 20 wherein said tomato plantlet is maintained at about 25° C. while being exposed to total darkness.

Claim 24

A method according to claim 15 wherein the excised tomato plant tissue of step (d) is excised tomato plant tissue selected from the group consisting of leaf tissue, stem tissue, cotyledon tissue and root tissue.

Claim 25

A method according to claim 15 wherein said pre-enzyme treatment (PET) solution of step (e) contains plant hormones.

Claim 26

A method according to claim 23 wherein said plant hormones are plant hormones that encourage cell growth and differentiation.

Claim 27

A method according to claim 23 wherein said plant hormones are plant hormones selected from the group consisting of auxins and cytokinins.

Claim 28

A method according to claim 15 wherein said excised tomato plant tissue in step (e) is treated with said "pre-enzyme treatment" (PET) solution for about 8-14 hours.

Claim 29

A method according to claim 15 wherein said "pre-enzyme treatment" (PET) solution of step (e) is chilled.

Claim 30

A method according to claim 27 wherein said chilled "pre-enzyme treatment" (PET) solution is chilled to about 10° C.

Claim 31

An in vitro method for preconditioning cultivated tomato plants that will be used as sources of protoplast donating plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured protoplasts, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from cultivated tomato plants, in sterilized water, at about 25° C., until a shoot extends from said seed, (b) growing said shoot from step (a) in a soil free nutrient medium, to which no exogenous plant hormones have been added, said shoot being grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark), until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness, for about 48 hours, while maintaining said tomato plantlet at about 25° C., (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with a chilled "pre-enzyme treatment" (PET) solution, for about 8 hours to about 14 hours, said "pre-enzyme treatment" (PET) solution being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, (f) using said excised, treated tomato plant tissue from step (e) as a source of protoplast donating tomato plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured tomato protoplasts.

Claim 32

An in vitro method for preconditioning cultivated tomato plants that will be used as sources of protoplast donating plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured protoplasts, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from cultivated tomato plants, in sterilized water, at about 25° C., until a shoot extends from said seed, (b) growing said shoot from step (a) in a soil free nutrient medium, to which no exogenous plant hormones have been added, said shoot being grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark), until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness, for about 48 hours, while maintaining said tomato plantlet at about 25° C., (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with a chilled "pre-enzyme treatment" (PET) solution, for about 8 hours to about 14 hours, said "pre-enzyme treatment" (PET) solution being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, (f) using said excised, treated tomato plant tissue from step (e) as a source of protoplast donating tomato plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured tomato protoplasts.

Claim 33

A method according to claim 29 wherein said cultivated tomato plants are Lycopersicon esculentum tomato plants.

Claim 34

A method according to claim 30 wherein said Lycopersicon esculentum tomato plants are Lycopersicon esculentum cultivars selected from the group comprised of Lycopersicon esculentum cv. Red Cherry, Lycopersicon esculentum cv. Cocktail Cherry, Lycopersicon esculentum cv. VFNT Cherry, Lycopersicon esculentum cv. VF36, Lycopersicon esculentum cv. Manapal, Lycopersicon esculentum cv. Floradade, Lycopersicon esculentum cv. UC82, Lycopersicon esculentum cv. Roma, Lycopersicon esculentum cv. Beefsteak, and Lycopersicon esculentum cv. San Marzano.

Claim 35

A method according to claim 29 wherein the excised tomato plant tissue of step (d) is excised tomato plant tissue selected from the group consisting of leaf tissue, stem tissue, cotyledon tissue and root tissue.

Claim 36

A method according to claim 29 wherein said chilled "pre-enzyme treatment" (PET) solution from step (e) is chilled to about 10° C.

Claim 37

An improved method for regenerating cultivated tomato plants from cultured tomato protoplasts, said regeneration method including the steps of: (1) isolating tomato protoplasts from protoplast donating tomato plant tissue derived from cultivated tomato plants, (2) culturing said protoplasts from step (1), in growth media containing plant hormones that encourage cell growth, until said protoplasts divide and form small multi-cellular colonies composed of about 100 protoplast-derived cells growing in close proximity to one another, said small multi-cellular colonies not being visible to the naked eye, (3) culturing said small multi-cellular colonies from step (2), in growth media containing plant hormones that encourage cell growth, until said small multi-cellular colonies from small mini-calli that are about 3-5 mm in diameter, and visible to the naked eye, (4) culturing said small mini-calli from step (3), in differentiation media containing plant hormones that encourage differentiation and further growth, for about one week, (5) culturing the calli from step (4) in differentiation media, containing plant hormones that encourage further differentiation and growth of said calli, until shoots grow from said calli, (6) removing said shoots from said shoot-bearing differentiated calli of step (5), (7) culturing said shoots from step (6), in growth media containing plant hormones that encourage root formation, until said shoots develop roots, and (8) growing said rooted shoots from step (7) until said rooted shoots become tomato plants; wherein the improvement comprises an in vitro method for preconditioning a protoplast donating cultivated tomato plant prior to protoplast isolation, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from a cultivated tomato plant, in sterilized water, at about 25° C., until a shoot extends from said seed, (b) growing said shoot from step (a) in a soil free nutrient medium, to which no exogenous plant hormones have been added, said shoot being grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark), until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness for about 48 hours, while maintaining said tomato plantlet at about 25° C., (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with a chilled "pre-enzyme treatment" (PET) solution, for about 8 hours to about 14 hours, said PET solution being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, (f) using said excised, treated tomato plant tissue from step (e) as protoplast donating tomato plant tissue for protoplast isolation.

Claim 38

An improved method for regenerating cultivated tomato plants from cultured tomato protoplasts, said regeneration method including the steps of: (1) isolating tomato protoplasts from protoplast donating tomato plant tissue derived from cultivated tomato plants, (2) culturing said protoplasts from step (1), in growth media containing plant hormones that encourage cell growth, until said protoplasts divide and form small multi-cellular colonies composed of about 100 protoplast-derived cells growing in close proximity to one another, said small multi-cellular colonies not being visible to the naked eye, (3) culturing said small multi-cellular colonies from step (2), in growth media containing plant hormones that encourage cell growth, until said small multi-cellular colonies from small mini-calli that are about 3-5 mm in diameter, and visible to the naked eye, (4) culturing said small mini-calli from step (3), in differentiation media containing plant hormones that encourage differentiation and further growth, for about one week, (5) culturing the calli from step (4) in differentiation media, containing plant hormones that encourage further differentiation and growth of said calli, until shoots grow from said calli, (6) removing said shoots from said shoot-bearing differentiated calli of step (5), (7) culturing said shoots from step (6), in growth media containing plant hormones that encourage root formation, until said shoots develop roots, and (8) growing said rooted shoots from step (7) until said rooted shoots become tomato plants; wherein the improvement comprises an in vitro method for preconditioning a protoplast donating cultivated tomato plant prior to protoplast isolation, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from a cultivated tomato plant, in sterilized water, at about 25° C., until a shoot extends from said seed, (b) growing said shoot from step (a) in a soil free nutrient medium, to which no exogenous plant hormones have been added, said shoot being grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark), until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness for about 48 hours, while maintaining said tomato plantlet at about 25° C., (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with a chilled "pre-enzyme treatment" (PET) solution, for about 8 hours to about 14 hours, said PET solution being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, (f) using said excised, treated tomato plant tissue from step (e) as protoplast donating tomato plant tissue for protoplast isolation.

Claim 39

A method according to claim 34 wherein said cultivated tomato plants are Lycopersicon esculentum tomato plants.

Claim 40

A method according to claim 34 wherein said Lycopersicon esculentum tomato plants are Lycopersicon esculentum cultivars selected from the group comprised of Lycopersicon esculentum cv. Red Cherry, Lycopersicon esculentum cv. Cocktail Cherry, Lycospersicon esculentum cv. VFNT Cherry, Lycopersicon esculentum cv. VF36, Lycopersicon esculentum cv. Manapal, Lycopersicon esculentum cv. Floradade, Lycopersicon esculentum cv. UC82, Lycopersicon esculentum cv. Roma, Lycopersicon esculentum cv. Beefsteak, and Lycopersicon esculentum cv. San Marzano.

Claim 41

A method according to claim 34 wherein the excised plant tissue in step (d) is excised plant tissue selected from the group consisting of leaf tissue, stem tissue, cotyledon tissue and root tissue.

Claim 42

A method according to claim 34 wherein said chilled "pre-enzyme treatment" (PET) solution from step (e) is chilled to about 10° C.

Claim 43

An in vitro method for preconditioning Lycopersicon esculentum tomato plants that will be used as sources of protoplast donating plant tissue when extracting protoplasts to be used for regenerating Lycopersicon esculentum tomato plants from cultured protoplasts, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from cultivated tomato plants, in sterilized water, at about 25° C., until a shoot extends from said seed, (b) growing said shoot from step (a) in a soil free nutrient medium, to which no exogenous plant hormones have been added, said shoot being grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark), until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness, for about 48 hours, while maintaining said tomato plantlet at about 25° C., (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with a chilled "pre-enzyme treatment" (PET) solution, for about 8 hours to about 14 hours, said "pre-enzyme treatment" (PET) solution being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, (f) using said excised, treated tomato plant tissue from step (e) as a source of protoplast donating tomato plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured tomato protoplasts.

Claim 44

An in vitro method for preconditioning Lycopersicon esculentum tomato plants that will be used as sources of protoplast donating plant tissue when extracting protoplasts to be used for regenerating Lycopersicon esculentum tomato plants from cultured protoplasts, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from cultivated tomato plants, in sterilized water, at about 25° C., until a shoot extends from said seed, (b) growing said shoot from step (a) in a soil free nutrient medium, to which no exogenous plant hormones have been added, said shoot being grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark), until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness, for about 48 hours, while maintaining said tomato plantlet at about 25° C., (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with a chilled "pre-enzyme treatment" (PET) solution, for about 8 hours to about 14 hours, said "pre-enzyme treatment" (PET) solution being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, (f) using said excised, treated tomato plant tissue from step (e) as a source of protoplast donating tomato plant tissue when extracting protoplasts to be used for regenerating cultivated tomato plants from cultured tomato protoplasts.

Claim 45

A method according to claim 39 wherein said Lycopersicon esculentum tomato plants are Lycopersicon esculentum cultivars selected from the group comprised of Lycopersicon esculentum cv. Red Cherry, Lycopersicon esculentum cv. Cocktail Cherry, Lycopersicon esculentum cv. VFNT Cherry, Lycopersicon esculentum cv. VF36, Lycopersicon esculentum cv. Manapal, Lycopersicon esculentum cv. Floradade, Lycopersicon esculentum cv. UC82, Lycopersicon esculentum cv. Roma, Lycopersicon esculentum cv. Beefsteak, and Lycopersicon esculentum cv. San Marzano.

Claim 46

A method according to claim 39 wherein the excised tomato plant tissue of step (d) is excised tomato plant tissue selected from the group consisting of leaf tissue, stem tissue, cotyledon tissue and root tissue.

Claim 47

A method according to claim 39 wherein said chilled "pre-enzyme treatment" (PET) solution from step (e) is chilled to about 10° C.

Claim 48

An improved method for regenerating Lycopersicon esculentum tomato plants from cultured tomato protoplasts, said regeneration method including the steps of: (1) isolating tomato protoplasts from protoplast donating tomato plant tissue derived from Lycopersicon esculentum tomato plants, (2) culturing said protoplasts from step (1), in growth media containing plant hormones that encourage cell growth, until said protoplasts divide and form small multi-cellular colonies composed of about 100 protoplast-derived cells growing in close proximity to one another, said small multi-cellular colonies not being visible to the naked eye, (3) culturing said small multi-cellular colonies from step (2), in growth media containing plant hormones that encourage cell growth, until said small multi-cellular colonies from small mini-calli that are about 3-5 mm in diameter, and visible to the naked eye, (4) culturing said small mini-calli from step (3), in differentiation media containing plant hormones that encourage differentiation and further growth, for about one week, (5) culturing the calli from step (4) in differentiation media, containing plant hormones that encourage further differentiation and growth of said calli, until shoots grow from said calli, (6) removing said shoots from said shoot-bearing differentiated calli of step (5), (7) culturing said shoots from step (6), in growth media containing plant hormones that encourage root formation, until said shoots develop roots, and (8) growing said rooted shoots from step (7) until said rooted shoots become tomato plants; wherein the improvement comprises an in vitro method for preconditioning a protoplast donating Lycopersicon esculentum tomato plant prior to protoplast isolation, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from a Lycopersicon esculentum tomato plant, in sterilized water, at about 25° C., until a shoot extends from said seed, (b) growing said shoot from step (a) in a soil free nutrient medium, to which no exogenous plant hormones have been added, said shoot being grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark), until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness for about 48 hours, while maintaining said tomato plantlet to about 25° C., (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with a chilled "pre-enzyme treatment" (PET) solution, for about 8 hours to about 14 hours, said PET solution being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, (f) using said excised, treated tomato plant tissue from step (e) as protoplast donating tomato plant tissue for protoplast isolation.

Claim 49

An improved method for regenerating Lycopersicon esculentum tomato plants from cultured tomato protoplasts, said regeneration method including the steps of: (1) isolating tomato protoplasts from protoplast donating tomato plant tissue derived from Lycopersicon esculentum tomato plants, (2) culturing said protoplasts from step (1), in growth media containing plant hormones that encourage cell growth, until said protoplasts divide and form small multi-cellular colonies composed of about 100 protoplast-derived cells growing in close proximity to one another, said small multi-cellular colonies not being visible to the naked eye, (3) culturing said small multi-cellular colonies from step (2), in growth media containing plant hormones that encourage cell growth, until said small multi-cellular colonies from small mini-calli that are about 3-5 mm in diameter, and visible to the naked eye, (4) culturing said small mini-calli from step (3), in differentiation media containing plant hormones that encourage differentiation and further growth, for about one week, (5) culturing the calli from step (4) in differentiation media, containing plant hormones that encourage further differentiation and growth of said calli, until shoots grow from said calli, (6) removing said shoots from said shoot-bearing differentiated calli of step (5), (7) culturing said shoots from step (6), in growth media containing plant hormones that encourage root formation, until said shoots develop roots, and (8) growing said rooted shoots from step (7) until said rooted shoots become tomato plants; wherein the improvement comprises an in vitro method for preconditioning a protoplast donating Lycopersicon esculentum tomato plant prior to protoplast isolation, said in vitro preconditioning method comprising: (a) germinating a surface sterilized protoplast donating tomato plant seed, derived from a Lycopersicon esculentum tomato plant, in sterilized water, at about 25° C., until a shoot extends from said seed, (b) growing said shoot from step (a) in a soil free nutrient medium, to which no exogenous plant hormones have been added, said shoot being grown at about 25° C., with a photoperiod of about 16 hr/8 hr (light/dark), until said shoot develops into a small tomato plantlet having expanded leaves and a substantial stem, (c) exposing said tomato plantlet from step (b) to total darkness for about 48 hours, while maintaining said tomato plantlet to about 25° C., (d) excising tissue from said tomato plantlet of step (c), (e) treating said excised tomato plant tissue of step (d) with a chilled "pre-enzyme treatment" (PET) solution, for about 8 hours to about 14 hours, said PET solution being comprised essentially of an osmoticum to which exogenous auxin and cytokinin plant hormones have been added, (f) using said excised, treated tomato plant tissue from step (e) as protoplast donating tomato plant tissue for protoplast isolation.

Claim 50

A method according to claim 43 wherein said Lycopersicon esculentum tomato plants are Lycopersicon esculentum cultivars selected from the group comprised of Lycopersicon esculentum cv. Red Cherry, Lycopersicon esculentum cv. Cocktail Cherry, Lycopersicon esculentum cv. VFNT Cherry, Lycopersicon esculentum cv. VF36, Lycopersicon esculentum cv. Manapal, Lycopersicon esculentum cv. Floradade, Lycopersicon esculentum cv. UC82, Lycopersicon esculentum cv. Roma, Lycopersicon esculentum cv. Beefsteak, and Lycopersicon esculentum cv. San Marzano.

Claim 51

A method according to claim 43 wherein the excised plant tissue in step (d) is excised plant tissue selected from the group consisting of leaf tissue, stem tissue, cotyledon tissue and root tissue.

Claim 52

A method according to claim 43 wherein said chilled "pre-enzyme treatment" (PET) solution from step (e) is chilled to about 10° C.