Clone of the sea kale (Crambe maritima L.), and process for vegetative propagation thereof

A method for the vegetative propagation of the sea kale species is described. Buds are obtained from in vitro neoformation on a callus, which in turn is obtained from fragments of petioles or leaf laminas of the plant. The rootless plants are then micropropogated in vitro in a plant culture medium and cytokinines in the absence of auxins. The rootless plants bearing one or two leaves are cultured in vitro in a plant growth broth and one or more auxins in the absence of cytokinins until roots develop. The rooted plants are transferred to a solid plant growth substrate and bred under conditions of a natural photoperiod of illumination until a plant is obtained which is capable of growth in the field.

The invention relates to a clone of the vegetable species CRAMBE which is 
not currently cultivated, obtained by neoformation in vitro on a callus of 
a petiole or lamina fragment removed from a plant of long-standing 
cultivation, and a method of vegetative propagation by micropropagation in 
vitro of the clone and the Crambe species. 
The clone of Crambe obtained constitutes a new vegetable for the farmer and 
the consumer. Vegetative propagation, which makes use of in vitro 
techniques, makes it possible to produce, on a large scale and very 
homogeneously, cuttings to be placed in the field, and to provide for 
rapid launching of this new production. 
DESCRIPTION 
The invention relates to a clone of sea kale originating from a plant 
maintained in a conservatory since the 19th century, a fragment of which 
was subjected to organogenesis by culturing it in vitro, and also to a 
process for vegetative propagation thereof in vitro, leading to the 
production of cuttings which can be used by farmers. 
The sea kale (Crambe maritima L.), which was probably cultivated to a small 
extent in France during the 19th century, has nowadays completely 
disappeared from the array of cultivated vegetables marketed in France, 
and in Europe. The description of this vegetable, as well as the main 
phytotechnical aspects of cultivating it, are described in several 
encyclopedic works on agronomy dating from the end of the 19th century 
(Vilmorin-Andrieux 1883, Nicholson 1895). In this plant species, which is 
found on sea coasts and is perennial by means of its rhizome, the young 
shoots in the etiolated state, on the resumption of growth in spring, form 
the edible organ. The use of root cuttings in the field is the method of 
propagation of the species and hence the traditional process for 
establishing a crop for this species. 
On leaving the Ecole Nationale Superieure d'Horticulture of Versailles, 
where he held a post until August 1971, the present inventor took a sample 
of a rhizome fragment from a sea kale plant present in the departmental 
collection of edible plants at this establishment since the last century. 
The maintenance of this plant by successive fragmentation of the rhizome 
throughout generations without there being any modification of the plant 
from the standpoint of genotype--the description of the plant conforming 
with the description given by the above-mentioned authors--enables the 
inventor to assert that the plant referred to above was cultivated during 
the last century in the state in which it existed at the time the inventor 
removed the sample. Hence it cannot be of the same type as the wild crambe 
which we encounter in resorts on the coast of Brittany or Normandy. Since 
that date, the plant has been maintained in the botanical conservatory of 
the Department of Vegetable Crops of the Ecole Nationale d'Ingenieurs des 
Travaux Agricoles, Horticulture Section, of Angers, where the inventor is 
currently working.

From this parent plant, the inventor has created a clone according to the 
following process: 
A fragment of an organ, which can be a petiole or lamina, is disinfected in 
a 10% strength NaOCl solution under vacuum for 10 minutes, rinsed in three 
washes of sterile water and cultured in test tubes in an air-conditioned 
room under the following conditions: 
Temperature: day=24.degree. C., night=18.degree. C. 
Light: 10,000 lux for 16 hours/24 hours. 
The culture medium used has the following composition (amounts expressed 
per liter of culture medium): 
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NH.sub.4 NO.sub.3 0.48 g 
Ca(NO.sub.3).sub.2.4H.sub.2 O 
0.71 g 
KNO.sub.3 0.15 g 
KCl 0.075 g 
MgSO.sub.4.7H.sub.2 O 
0.493 g 
(NH.sub.4).sub.2 SO.sub.4 
0.265 g 
KH.sub.2 PO.sub.4 0.140 g 
K.sub.2 HPO.sub.4 0.175 g 
Fe EDTA(*) 5 ml of stock solution 
H.sub.3 BO.sub.3 2.48 mg 
MnSO.sub.4 3.38 mg 
ZnSO.sub.4 1.15 mg 
KI 0.30 mg 
Na.sub.2 MoO.sub.4 
0.10 mg 
CuSO.sub.4 0.05 
CoSO.sub.4 0.05 mg 
NiSO.sub.4 0.05 mg 
AlCl.sub.3 0.05 mg 
Glutamine 200 mg 
Adenine 40 mg 
Sucrose 30 g 
Agar C 8 g 
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(*)The stock solution of Fe EDTA (iron ethylenediaminetetraacetic acid) 
is prepared in the following manner: 557 mg of FeSO.sub.4.7H.sub.2 O are 
mixed with 750 mg of Na.sub.2 EDTA, and the volume of the mixture is 
adjusted to 100 ml with distilled water. 
The pH of the above culture medium is adjusted to 5.6 with 1/10 HCl 
solution. 
From these explants, calluses develop if an auxin, which can be indolacetic 
acid (0.2 to 0.7 mg/l, preferably 0.5 mg/l), and a mixture of cytokinins 
are added to the above medium in a suitable concentration ratio, for 
example, an auxin/cytokinin concentration ratio, expressed in mg/liter, of 
0.066, the mixture of cytokinins also being in a suitable ratio, this 
mixture of cytokinins possibly consisting of, for example, kinetin (6 to 8 
mg/l) and 6-benzylaminopurine (1 to 2 mg/l, preferably 1.5 mg/l), these 
two cytokinins also being in a suitable ratio, for example, in a 
concentration ratio (mg/l) of 4. 
Under these conditions, organogenesis takes place in 20-28 days: there is 
neoformation of several buds on the calluses, and the buds then develop 
leaves. Each bud is then separated and then pricked out onto a so-called 
propagation medium according to the process explained below, which is also 
included in the present invention. The totality of the plants thus 
obtained, which have undergone subsequent pricking out onto a suitable 
rooting medium--which process also forms part of the invention--is 
homogeneous and formed of plants which are all identical to each other: 
this hence constitutes a clone of seal kale obtained according to the 
process explained above. 
The characteristics of the plants of the clone thus obtained are collated 
below: 
From the morphological standpoint, the plants of the clone possess leaves 
with a flattened lamina, the marginal notches of which, known as teeth and 
few in number, have a constant which is characteristic of all the plants 
in the clone and which can be designated by an index I defined as follows: 
##EQU1## 
I.s=length, expressed in cm, of the internerve space in question, 
enclosing a sinus (position of this space identical for all plants 
studied). 
N=number of teeth present in the internerve space (I.s) in question. 
L=total length of the lamina of the leaf, expressed in cm. 
This index settles at an average value of 0.068. 
The morphological homogeneity of the plants which form the clone created by 
the process described above is verified in terms of the index I by 
statistical analysis (determination of the standard deviation), as seen 
from the results which appear in Table I below: 
TABLE I 
______________________________________ 
Plants L (cm) I.s (cm) N I 
______________________________________ 
1 24.9 5.1 6 0.047 
2 31.6 9.1 20 0.069 
3 37.5 12.5 40 0.085 
4 31.9 8.9 24 0.084 
5 39.5 7.4 20 0.068 
6 34.4 5.5 12 0.063 
7 37.5 9.7 19 0.052 
8 23.0 5.5 10 0.079 
9 18.5 3.2 4 0.067 
Average 0.068 
Standard deviation 
0.014 
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Furthermore, the leaves (lamina+petiole) of the plants of the clone reach, 
when fully developed, an average length of 48 cm and are green due to the 
absence of anthocyanine pigments. Finally, for a suitable cultivation 
site, at whatever date they are planted, the plants originating from 
culture in vitro, according to the process which also forms part of the 
present invention, do not exhibit the floral state during the year when 
these plants were set up in the field after the micro-propagation phase in 
vitro. When they resume growth in situ or in an air-conditioned chamber, 
the plants of the clone, after the first year of growth, develop, in 
darkness, shoots--the edible organ of the plant--which are of culinary 
value. 
The invention also relates to a process for propagating the clone of sea 
kale which leads to the production of cuttings which can be used directly 
by farmers. The process can also be used on the sea kale species. 
The buds originating from organogenesis in vitro, following the method 
according to the present invention described above, are separated from the 
callus under sterile conditions and then pricked out in a glass bowl 
containing 130 ml of a medium identical to the neoformation medium 
specified above except in respect of the nature of the growth substances 
and their balance: the mixture contains exclusively a cytokinin (e.g., 
6-benzylaminopurine in a concentration of 6 to 8 mg/l, preferably 7 mg/l) 
or a mixture of cytokinins in a suitable ratio, for example, in a 
concentration ratio, expressed in mg/liter, of 1.66, this mixture of 
cytokinins possibly being kinetin (4 to 6 mg/l, preferably 5 mg/l) and 
6-benzylaminopurine (2 to 4 mg/l, preferably 3 mg/l). The climatic 
conditions relating to the neoformation process are maintained in the 
present process. When all the conditions are observed, the buds induce 
lateral buds, the average number of which is 5, and which develop in 3 
weeks, at the end of which a further pricking out will be necessary, with 
separation of the neoformed buds. This propagation process in vitro, which 
is also a subject of the present invention, enables 18,125 cuttings to be 
obtained after 18 weeks, starting from one neoformed bud. 
When the requisite number of cutting-plants to be cultivated in the field 
is potentially reached, the buds are pricked out in vitro, in test tubes 
or in a glass bowl, on a so-called rooting medium the composition of which 
is identical to that of the neoformation or propagation medium with the 
exception of the growth substances which, in the present case, are 
represented exclusively by one or more auxins (e.g., naphthalene acetic 
acid in a concentration of, for example, 0.1 mg/l). The climatic 
conditions, which remain identical to the previous conditions, enable 3 to 
4 white roots to be developed after 2 to 3 weeks. 
The young plants thus obtained, equipped with 2 to 4 cm roots, are 
transferred to pots containing a non-sterilized, moistened substrate, this 
substrate possibly being composed of, for example, one-half yellow peat 
and one-half sand, and moistened to 80% of its water retention capacity. 
The pots are placed in mini-greenhouses equipped with an adjustable cover, 
under the following climatic conditions: 
Day temperature: 20.degree. C. 
Night temperature: 15.degree. C. 
Natural illumination of the place where the intensity was maintained at 
less than 15,000 lux. 
Maintained thus under these conditions, the plants develop both as regards 
their foliage (lamina approximately 10 cm for the oldest leaves) and as 
regards their root system (emission of three or four long, slightly 
tuberous roots, on which a fine and abundant lateral root-hair growth 
appears). After a 4 to 6 week breeding period, the plants can be 
transferred to the production field to set up production crops of the sea 
kale vegetable. 
With the sea kale, the main advantages of vegetative micropropagation in 
vitro, compared to traditional propagation by root cuttings, are as 
follows: 
complete homogeneity of the cuttings to be placed in the field, leading to 
very great homogeneity of the field crop, 
the propagation of an individual belonging to the clone can be carried out 
continuously, independently of the season and the climatic conditions, the 
micropropagation in vitro of sea kale makes it possible to increase 
dramatically the potential for production of cuttings starting from one 
individual, 
finally, it makes it possible to ensure for rapid launching of this new 
production by farmers. 
FIGS. 1 and 2 illustrate plants obtained according to the invention.