Abstract:
A tissue culture technique and a culture medium used therewith for asexually propagating a plurality of plants of the family Cruciferae are disclosed. 
     According to one aspect of the invention, an excised portion of a donor plant is placed in a culture medium comprising organic and inorganic salts and a preferable concentration of 1 mg/l of Indoleacetic Acid, 0.5 mg/l of Kinetin and 40 mg/l of Adenine Sulfate. The medium induces the simultaneous initiation of buds and roots by the explant to form a plurality of plantlets which may be transplanted into soil for the timely initiation of food and medicinal crops. 
     In accordance with a second aspect of the invention, the pre-culture storage of the requisite plant part in a convenient, disease-free, and pest-free manner is disclosed whereby callus from the excised portion of the donor plant is maintained on a first medium for subsequent transfer to the medium described above. The callus is initiated and maintained, for periods in the order of a year or more, on a medium containing organic and inorganic salts and preferable concentrations of 1.86 mg/l of naphthaleneacetic acid and 2.25 mg/l of N 6  -Benzyladenine.

Description:
FIELD OF THE INVENTION 
     This invention relates generally to a tissue culture method for the asexual propagation of plants and to a culture medium for use in the tissue culture process. 
     The propagation of plants through seeds has always suffered a number of limitations. The plants thus propagated may be quite variable, since they do not necessarily contain all the parental characteristics, and a significant proportion of a crop may consequently be of an unsalable quality. Additionally, proper germination of the seeds is dependent upon, and influenced by, such external conditions as weather, the nature and pH of the soil, organisms in the soil, the availability of water, temperature, light and a loss of vigor during storage of the seeds. The number of seed-propagated plants available for consumption is limited both by the seasonal availability of the crop, seed supply, and the amount of growing space. 
     Recent forecasts predicting an increasing food shortage in the world have stimulated efforts to discover more efficient and reliable methods for food production. In recent years, plant tissue culture methods have gained importance as a means for vegetative propagation of food, ornamental and medicinal crops owing to the ability of such a technique to produce rapid multiplication and proliferation of genetically uniform plants, thereby assuring that the desired characteristics of the selected donor plants are retained. 
     In the tissue culture technique, a piece of a donor plant is excised and placed in a culture medium comprising nutrients in the form of organic and inorganic compounds and hormones. Because the size of the excised plant part is generally in the order of two or three millimeters, the number of cultures which may be induced from a single donor plant may greatly exceed the number of seeds such a plant would ordinarily produce. The rate of multiplication thus attainable is even further enhanced by the fact that one need not wait for the plant to produce seeds but may excise plant parts at virtually any time during the plant&#39;s growth. Accordingly, the tissue culture technique enables timely increases of plant stock and hastens the availability of new varieties. Finally, the minimal size of the excised plant part lends itself to convenient storage under sterile conditions so that pest and plant disease problems conventionally associated with the maintenance of stock plants is virtually eliminated. 
     Conventionally, tissue culture techniques have been tailored to the stages of growth for the particular plant involved. These growth stages may be described as the induction stage, where the explant is established aseptically on the medium and allowed to respond to the medium via rapid growth and enlargement; the multiplication stage, wherein the explant produces a mass of undifferentiated cells called callus; the differentiation stage wherein the cells give rise to shoots and/or roots to form a plurality of separate plantlets; and the hardening stage wherein the plantlets are separated and allowed to develop from the heterotropic to the autotropic stage. During the induction stage, the explants have conventionally been placed in a nutritive medium containing organic and inorganic salts and growth regulators or growth hormones. 
     In an article entitled &#34;Chemical Control of Adventitious Organ Formation in Lactuca Sativa Explants&#34; by M. R. Doerschug, et al. (Amer. J. Bot. 54 (4): 410-413 (1967)), for example, kinetin (6-furfurylaminopurine) and adenine (6-aminopurine) are shown to promote the initiation of adventitious buds in excised cotyledons of Lactuca Sativa. A medium containing inorganic and organic salts, indoleacetic acid (IAA) and either kinetin or adenine were utilized to induce bud formation from cotyledon explants. 
     It is an object of the invention to promote a rapid proliferation of simultaneous shoot and root formation on a single culture medium. 
     It is another object of the invention to bring about rapid and multiple propagation of plants from the explant of a selected donor. 
     The culture technique and culture medium described herein are particularly suited for plants of the family Cruciferae. The technique basically comprises the steps of excising a part of the donor plant, culturing the excised part, hereinafter referred to as the explant, on a medium containing IAA, kinetin and a salt of adenine to induce differentiation of the explant into a plurality of plantlets, and transplanting the plantlets into soil. 
     The culture medium described herein promotes the simultaneous formation by the explant of shoots and roots so that rapid plant proliferation is achieved. 
     The disclosed technique and culture medium additionally lend themselves to a continuous or batch process whereby callus from the explant may be conveniently stored for a period in the order of a year or more and withdrawn as needed when required for propagation of a large quantity of plantlets so that plants may be timely grown therefrom as the need arises. 
     Further aspects and advantages of the invention will be apparent from the following description of the preferred embodiment. 
    
    
     BRIEF DESCRIPTION OF THE DRAWING 
     The sole FIGURE herein is a graphic representation of explant growth on the culture medium. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The technique disclosed herein, as well as the culture medium used therewith, is particularly suited for plants of the family Cruciferae and basically comprises the steps of excising a part of the donor plant, culturing the excised part on a medium containing IAA, kinetin and a salt of adenine to induce differentiation of the explant into a plurality of plantlets, and the subsequent transplanting of the plantlets into the soil. As will be discussed in greater detail, the medium promotes the simultaneous formation of shoots and roots which form the plantlets and enables the plantlets to quickly transform to an autotropic state. 
     In accordance with one aspect of the invention, and as supported in the examples set forth below, an excised part of a donor plant is placed in a tissue culture medium hereinafter referred to as Medium A and containing the following nutrients of organic and inorganic salts and growth regulators: 
     
         ______________________________________INORGANIC SALTS  (mg/l)______________________________________KH.sub.2 PO.sub.4            300KNO.sub.3        1000NH.sub.4 NO.sub.3            1000Ca(NO.sub.3).sub.2 . 4H.sub.2 O            500MgSO.sub. 4 . 7H.sub.2 O            71.6KCl              65MnSO.sub.4 . H.sub.2 O            4.92ZnSO.sub.4 . 7H.sub.2 O            2.67H.sub.3 BO.sub.3 1.6KI               0.8NaEDTA           37.2FeSO.sub.4 . 7H.sub.2 O            27.8ORGANIC SALTS    (mg/l)______________________________________Thiamine . HCl   0.1Nicotinic Acid   0.5Pyridoxine . HCl 0.1My0-inositol     100Glycine          2Sucrose          30,000Agar             10,000GROWTH REGULATORS            (mg/l)______________________________________Indoleacetic Acid            1(IAA)kinetin          0.5adenine sulfate  40______________________________________ 
    
     The above-described composition initiates the simultaneous formation by the excised tissue of shoots and roots to form a plurality of plantlets in the culture medium during a period of approximately 14 to 30 days. 
     In accordance with a second aspect of the invention, the excised part of the donor plant is first placed in another medium hereinafter referred to as Medium 1. 
     Medium 1 is utilized to induce and maintain callus formation on the excised plant part. The callus may be stored in this manner for a period in the order of a year or more until a need for duplicates of the donor plant arises, whereupon a portion of the callus is excised and placed on Medium A. Medium 1 contains the inorganic and organic salts, and the amounts therefor, indicated above. The growth regulators utilized is this medium, however, are: 
     1.86 mg./l of Naphthaleneacetic acid 
     2.25 mg/l N 6  -Benzyladenine (N 6  BA) 
     Further details and modifications to the preferred embodiment of the subject invention will become apparent hereinbelow wherein the utility of the present invention on various varieties of Cruciferae is demonstrated. 
     In ascertaining the preferred tissue culture method for asexually reproducing species of Cruciferae, a large number of experiments were performed with lettuce of the `Black Seeded Simpson` and `Summer Bibb` varieties. 
     Lettuce seeds were sterilized for five minutes in a solution of Chlorox, (a trademark owned by The Chlorox Company for bleach) and rinsed several times with sterile distilled water. The seeds were then placed in sterilized petri dishes containing filter paper and distilled water and allowed to germinate in the dark for 4 to 5 days. After this period, the seedlings were dissected for various plant parts under aseptic conditions. These excised parts included the cotyledon, epicotyl, hypocotyl, plumule and radicle. In general, the size of the explant varied from 1mm to 3mm. 
     Additionally, a head of lettuce of another variety obtained from a supermarket was used in these studies by excising 2-3mm pieces from the innermost leaves near the apical meristem. 
     All the plant parts thus obtained were planted onto the Medium 1 for callus induction and maintenance or onto Medium A for shoot and root differentiation. The cultures on Medium 1  were maintained under dark conditions for up to six months and portions of the callus subsequently excised and transferred to Medium A. The cultures on Medium A were exposed to GroLux Lights for 30 days with a 16 hour daily light schedule at an intensity at 360μW/cm 2 . (GroLux is a trademark owned by GTE Sylvania for a lamp having a spectral output matched to the absorption spectrum of Chlorophyll-A). 
     In accordance with the invention, the same technique was utilized whether the culture contained callus or the actual plant part. A comparison of results obtained for the various plant parts as indicated in Table 1 below shows that optimum organogenesis was derived from the cotyledon and leaf. 
     
                                           TABLE I__________________________________________________________________________Growth and Organogenesis of Various Seedlingparts and entire plant parts on medium__________________________________________________________________________                      No. of Shoots/Explant  Rate of Organo-            Time to Organo-                      after 30 daysPlant Part  genesis   genesis (Days)                      Var. `Simpson.`                              Var. `Summer Bibb`__________________________________________________________________________Cotyledon  30/30     10        10      20Epicotyl  30/30     15        6       11Hypocotyl  27/30     20        8       13Plumule  25/30     18        5       8Radicle  20/30     30        5       5Leaf   30/30     10        12      17__________________________________________________________________________ *indicates the number of differentiating explants/number of explants cultured. 
    
     Having thus determined that the cotyledon explants yielded optimum results, that part was utilized in the remaining tests. 
     The effect upon shoot, leaf, bud and root formation caused by variations in the concentrations of the three growth regulators IAA, kinetin and adenine sulfate was investigated next by utilizing culture media with varying concentrations of each and examining the plantlets after a 30 day period. Tables 2a-b, 3a-b and 4a-b show the effects of singularly varying the concentration for culture of &#34;Summer Bibb&#34; and &#34;Simpson&#34; explants. Similarly, Tables 5a-c, 6a-c and 7a-c  demonstrate the effects on &#34;Summer Bibb&#34; of varying two of the three regulator concentrations simultaneously. It may be observed from these tables that optimum results were obtained when the culture medium included 1 mg/l of IAA, 0.5 mg/l of kinetin and either 20 or 40 mg/l of adenine sulfate. 
     
                                           TABLE 2A__________________________________________________________________________(BLACK SEEDED SIMPSON)__________________________________________________________________________0.5 mg/l Kinetin40 mg/l Adenine     EXPLANTS            NO. WITH                  NO. OF                       NO. WITH                             NO. OF                                  NO. WITH                                        NO. OFSulfate   CULTIVATED            BUDS  BUDS SHOOTS                             SHOOTS                                  ROOTS ROOTS__________________________________________________________________________+0.5 mg/l IAA     20     20    M    20    8    13    5+1 mg/l IAA     20     20    H    20    12   20    8+5 mg/l IAA     20     20    M    16    5    12    6+10 mg/l IAA     20     16    M    7     2    9     4+20 mg/l IAA     20     13    M    2     1    8     4+50 mg/l IAA     20     6     S    1     1    5     3+100 mg/l IAA     20     2     S    --    --   --    --__________________________________________________________________________ S = 1-2 buds M = 3-8 buds H = more than 8 buds 
    
     
                                           TABLE 2B__________________________________________________________________________(SUMMER BIBB)__________________________________________________________________________0.5 mg/l Kinetin40 mg/l Adenine     PLANT PARTS             NO. WITH                   NO. OF                        NO. WITH                              NO. OF                                   NO. WITH                                         NO. OFSulfate   CULTIVATED             BUDS  BUDS SHOOTS                              SHOOTS                                   ROOTS ROOTS__________________________________________________________________________+0.5 mg/l IAA     20      20    M    20    8    11    2+1 mg/l IAA     20      20    H    20    20   14    4+5 mg/l IAA     20      20    M    15    7    12    3+10 mg/l IAA     20      16    M    8     2    12    2+20 mg/l IAA     20      13    M    5     2    10    2+50 mg/l IAA     20      6     S    1     1    5     1+100mg/l IAA     20      2     S    --    --   --    --__________________________________________________________________________ S = 1-2 buds M = 3-8 buds H = more than 8 buds 
    
     
                                           TABLE 3A__________________________________________________________________________BLACK SEEDED SIMPSON__________________________________________________________________________1.0 mg/l IAA40 mg/l Adenine     NO. OF PLANT              NO. WITH                    NO. OF                         NO. WITH                               NO. OF                                    NO. WITH                                          NO. OFSulfate   PARTS CULTURED              BUDS  BUDS SHOOTS                               SHOOTS                                    ROOTS ROOTS__________________________________________________________________________ +0.1 mg/lKinetin   20       17    M    16    7    16    7 +0.5 mg/lKinetin   20       20    H    20    12   20    5 +1 mg/lKinetin   20       20    H    20    9    18    2 +5 mg/lKinetin   20       13    M    10    3    --    -- +10 mg/lKinetin   20       7     S    2     1    --    -- +50 mg/lKinetin   20       3     S    --    --   --    --__________________________________________________________________________ S = 1-2 buds M = 3-8 buds H = more than 8 buds 
    
     
                                           TABLE 3B__________________________________________________________________________SUMMER BIBB__________________________________________________________________________1.0 mg/l IAA40 mg/l Adenine     NO. OF PLANT              NO. WITH                    NO. OF                         NO. WITH                               NO. OF                                    NO. WITH                                          NO. OFSulfate   PARTS CULTURED              BUDS  BUDS SHOOTS                               SHOOTS                                    ROOTS ROOTS__________________________________________________________________________ +0.1 mg/lKinetin   20       18    M    18    10   18    6 +0.5 mg/lKinetin   20       20    H    20    20   20    4 +1 mg/lKinetin   20       20    H    20    14   20    2 +5 mg/lKinetin   20       15    M    11    2    --    -- +10 mg/lKinetin   20       9     S    4     1    --    -- +50 mg/lKinetin   20       4     S    --    --   --    --__________________________________________________________________________ S = 1-2 buds M = 3-8 buds H = more than 8 buds 
    
     
                                           TABLE 4A__________________________________________________________________________ADENINE SULFATE VARIATION                   (BLACK SEEDED SIMPSON)__________________________________________________________________________     NO. OF1.0 mg/l IAA     PLANT PARTS             NO. WITH                   NO. OF                        NO. WITH                              NO. OF                                   NO. WITH                                         NO. OF0.5 mg/l Kinetin     CULTURED             BUDS  BUDS SHOOTS                              SHOOTS                                   ROOTS ROOTS__________________________________________________________________________ +5 mg/ladenine sulfate     20      18    M    13    6    6     5 +10 mg/ladenine sulfate     20      19    M    15    7    10    8 +20 mg/ladenine sulfate     20      19    H    19    9    13    7 +40 mg/ladenine sulfate     20      20    H    20    12   17    6 +80 mg/ladenine sulfate     20      20    H    17    8    5     2 +150 mg/ladenine sulfate     20      12    M    6     3    3     1 +200 mg/ladenine sulfate     20      8     M    3     2    1     1__________________________________________________________________________ S = 1-2 buds M = 3-8 buds H = more than 8 buds 
    
     
                                           TABLE 4B__________________________________________________________________________ADENINE SULFATE VARIATION                   (SUMMER BIBB)__________________________________________________________________________     NO. OF1.0 mg/l IAA     PLANT PARTS             NO. WITH                   NO. OF                        NO. WITH                              NO. OF                                   NO. WITH                                         NO. OF0.5 mg/l KINETIN     CULTURED             BUDS  BUDS SHOOTS                              SHOOTS                                   ROOTS ROOTS__________________________________________________________________________ +5 mg/ladenine sulfate     20      18    M    17    6    6     2 +10 mg/ladenine sulfate     20      19    M    19    8    8     2 +20 mg/ladenine sulfate     20      19    H    19    15   10    3 +mg/ladenine sulfate     20      20    H    20    20   13    4 +80 mg/ladenine sulfate     20      20    H    18    14   7     2 +150 mg/ladenine sulfate     20      12    M    11    2    3     2 +200 mg/ladenine sulfate     20      8     M    7     1    1     1__________________________________________________________________________ S = 1-2 buds M = 3-8 buds H = more than 8 buds 
    
     
                       TABLE 5A______________________________________`VARIETY SUMMER BIBB`AVERAGE NUMBER OF SHOOTS FORMED PER EXPLANTConcentration IAA = 0.1 mg/l______________________________________ADENINE SULFATE          KINETIN (mg/l)(mg/l)           0.1     0.5     5     10______________________________________ 5               2.6     4.0     --    --10               3.0     4.8     --    --20               3.4     7.6     --    --40               3.2     7.2     --    --80               2.0     3.8     --    --______________________________________ 
    
     
                       TABLE 5B______________________________________`VARIETY SUMMER BIBB`AVERAGE NUMBER OF LEAVES FORMED PER EXPLANTIAA = 0.1 mg/l______________________________________ADENINE SULFATE          KINETIN (mg/l)(mg/l)           0.1     0.5     5     10______________________________________ 5               5.4     7.6     --    --10               6.2     9.8     --    --20               7.0     15.6    --    --40               6.2     13.2    --    --80               4.0     7.6     --    --______________________________________ 
    
     
                       TABLE 5C______________________________________`VARIETY SUMMER BIBB`AVERAGE NUMBER OF ROOTS FORMED PER EXPLANTIAA = 0.1 mg/l______________________________________ADENINE SULFATE          KINETIN (mg/l)(mg/l)           0.1     0.5     5     10______________________________________ 5               1.2     0.8     --    --10               1.3     1.0     --    --20               1.8     1.2     --    --40               1.3     1.2     --    --80               1.2     0.6     --    --______________________________________ 
    
     
                       TABLE 6A______________________________________`VARIETY SUMMER BIBB`AVERAGE NUMBEROF SHOOTS FORMED PER EXPLANTIAA = 1.0 mg/l______________________________________ADENINE SULFATE         KINETIN (mg/l)(mg/l)        0.1      0.5      5      10______________________________________ 5            3.6      7.0      2.2    210            4.8      7.8      1.8    1.620            8.2      12.8     1.4    1.040            11.6     20       0.6    --80            6        7.2      --     --______________________________________ 
    
     
                       TABLE 6B______________________________________`VARIETY SUMMER BIBB`AVERAGE NUMBEROF LEAVES FORMED PER EXPLANTIAA = 1.0 mg/l______________________________________ADENINE SULFATE         KINETIN (mg/l)(mg/l)        0.1      0.5      5      10______________________________________ 5            9.6      15.2     4.4    4.110            9.6      17.2     3.8    3.420            16.8     24.2     2.8    2.240            21.6     37.2     1.4    --80            12.2     15.8     --     --______________________________________ 
    
     
                       TABLE 6C______________________________________`VARIETY SUMMER BIBB`AVERAGE NUMBEROF ROOTS FORMED PER EXPLANTIAA = 1.0 mg/l______________________________________ADENINE SULFATE         KINETIN (mg/l)(mg/l)        0.1      0.5      5      10______________________________________ 5            2.8      1.4      0.6    0.410            3.0      1.8      0.4    0.120            3.2      2.0      --     --40            4.4      2.4      --     --80            2.2      1.4      --     --______________________________________ 
    
     
                       TABLE 7A______________________________________`VARIETY SUMMER BIBB`AVERAGE NUMBEROF SHOOTS FORMED PER EXPLANTIAA = 10 mg/l______________________________________ADENINE SULFATE         KINETIN (mg/l)(mg/1)        0.1      0.5      5      10______________________________________ 5            1.4      1.8      2.2    1.210            1.2      2.2      1.8    0.820            1.0      1.2      1.4    0.640            0.8      0.8      1.2    0.680            0.2      0.4      0.4    0.2______________________________________ 
    
     
                       TABLE 7B______________________________________AVERAGE NUMBEROF LEAVES FORMED PER EXPLANTIAA = 10 mg/l`VARIETY SUMMER BIBB`______________________________________ADENINE SULFATE         KINETIN (mg/l)(mg/l)        0.1      0.5      5      10______________________________________ 5            3        3.6      4.8    2.210            2        4.4      2.6    2.020            1.4      2.8      2.4    1.240            1.4      1.6      2.6    1.280            0.6      1.0      0.4    0.4______________________________________ 
    
     
                       TABLE 7C______________________________________AVERAGE NUMBEROF ROOTS FORMED PER EXPLANTIAA = 10 mg/l`VARIETY SUMMER BIBB`______________________________________ADENINE SULFATE         KINETIN (mg/l)(mg/l)        0.1      0.5      5      10______________________________________ 5            1.4      1.0      0.8    0.410            1.4      0.8      0.8    0.420            1.2      0.8      0.6    0.240            0.6      0.4      0.4    --80            0.4      0.4      0.2    --______________________________________ 
    
     The preferred age of the donor plant at the time of excision was determined by excising the cotyledon from donor plants having ages shown in Table 8 and culturing them in a medium comprising 1 mg/l IAA, 0.5 mg/l kinetin and 40 mg/l of adenine sulfate. As shown in Table 8, optimum shoot and root formation occurred for cotyledons excised from 4-day old donor plant. 
     
                       TABLE 8______________________________________THE EFFECT OF LETTUCE COTYLEDONEXPLANT AGE ON ORGANOGENESIS`VARIETY BLACK SEEDED SIMPSON`______________________________________EXPLANT AGE NO. OF     NO. OF     NO. OF(DAYS)      SHOOTS     LEAVES     ROOTS______________________________________2           8.89       34.13      0.914           9.14       36.00      1.868           7.66       33.73      0.5311          5.37       13.59      0.6616          3.88       8.44       0.2922          2.66       8.04       --25          2.82       6.62       --28          2.53       3.14       --______________________________________ 
    
     The period for which the explants should be maintained on Medium A prior to transplanting was determined by placing cotyledon explants on the culture medium and subjecting them to 360 μW/cm 2  of broad spectral light for 16 hours per day. From the FIGURE, it may be seen that one may obtain maximum roots, shoot and leaf formation at approximately the 40th day and that the increase in shoot, root and leaf formation is at a maximum between the 30th and 40th days. 
     In addition to lettuce, radish, beet, may apple (podophyllum peltatum), and chrysanthemum have been successfully propagated utilizing the method and media describe above. Table VI below summarizes the results obtained with a medium comprising 1 mg/l of IAA, 0.5 mg/l kinetin and 40 mg/l adenine sulfate. 
     
         ______________________________________                  #SHOOTS/   #ROOTS/PLANT     EXCISED PART EXPLANT    EXPLANT______________________________________Radish    cotyledon    10-15       7-10Beet      cotyledon    12-15      10-12May Apple Rhizome      4-5        6-8Chrysanthemum     Shoot tip with                   8-10      4-6     two pairs of     leaf primordia______________________________________