[P-052]
A COMPARATIVE MORPHOLOGICAL STUDY OF SOME VARIETIES OF Hyssopus officinalis L. TREATED WITH GAMMA RADIATION

Elvira GILLE and Florin FLORIA
"Stejarul" Research Centre, Alexandru cel Bun 6, 5600 Piatra Neamt - Romania

ABSTRACT

The treatment with gamma rays (2-5 kR) increased the genetic variability of Hyssopus officinalis plants in generation M1. The registered biological effects were differentiated by the doses used. The increase of the irradiation dose generally inhibited plant growth and development, seed germination, plant survival, compared to the local De Ciorani population. Exceptions are the variants treated with 5 kR, which presented positive effects in the behaviour of the investigated morphological parameters. The same great variability characterises the investigated plants from the point of view of the essential oil content. Gamma rays stimulated the biosynthesis of active principles of Hyssopi herba compared to the original population.

Our study did not show any positive relation between the morphological characters and the essential oil content.


INTRODUCTION

Hyssopus officinalis L. is a perennial plant cultivated for its essential oils. Being also a mellifera plant in food industry and in cosmetics (2,12).

The product used Hyssopi herba contains 0.1-1.4% essential oil with the main compounds: pinocamphone, isopinocamphone; a-pinene, b-pinene, camphene, a-terpinene, pinocampheol, 1,8-cineole, linalool, terpineol, and other one. The essential oil may be adulterated with spike, lavandin, lavender, rosemary, and camphor oils (3,5,8,10,11).

Due to its rich composition the plant has a series of properties as diuretic, depurative, carminative, spasmolithic, diaphoretic (1,3,4), emmenagogue, expectorant, stimulant, stomachic, and tonic. It is used in the treatment of asthma, rheumatism, sore throats, wounds, ulcers and tumors (6,7,10,12,13).

Our investigations aimed to improve the genetic variability of Hyssopus officinalis through experimental mutagenesis.


MATERIAL AND METHOD

Seed of Hyssopus officinalis, belonging the local population De Ciorani (pink flowers), from SCPMA - Fundulea, were gamma irradiated (121.000 R/h) and we obtained the following experimental variants: the control (De Ciorani population); 5 kR gamma rays; 15 kR gamma rays; 25 kR gamma rays. These were sown early in spring on an experimental field in Piatra Neamt. The plants were analysed in M1, I - III vegetation years (1997 - 1999). In the flowering phenophasa, we harvested sample (Hyssopi herba) for phytochemical analyses. The essential oils were extracted by hydrodistillation in a Neo-Clevenger apparatus and the quantity was expressed in g/100 g dry weight.

The analysed parameters were: plant height, number of floriferous stems (the second branching order) bush diameter, inflorescence branching number, length of the main inflorescence, number of floral circles/main inflorescence, number of flowers main inflorescence. The results were statistically interpreted.


RESULTS AND DISCUSSIONS

The hyssop population we studied proved a raised radiosensibility. Although we used equivalents of seeds, the number of individuals that germinated, grew and survived diminished drastically with the increasing of the irradiation dose, for example, using 25 kR only 8 and 54 at 5 kR compared to 83 individuals at the control. These aspects suggest same genetic effects, with physiological and biochemical repercussions: increase of lethality and reduction of the surviving ratio (6,7).


Table 1. Variation of some morphological parameters of Hyssopus officinalis L. variants treated with
gamma rays, in M1 generation (1st year of vegetation)
Mutagen treatment
Plant height (cm)
No of branches/plant
No. of inflorescences/branch 
Mean
s%
C=100
Mean
s%
C=100
Mean
s%
C=100
Control 
5 kR
15 kR
25 kR
40.6
38.2
32.3
21.9
35
41
62
58
100
94
80
54
32.6
12.4
12.7
4.2
45
44
43
45
100
89
99
52
4.5
4.1
4.5
2.4
45
43
40
31
100
38
39
8

Table 2. Some feature of Hyssopus officinalis L. inflorescences of gamma rays
treated plants, in M1 generation (1st year of vegetation)
Mutagen treatment
Principal inflorescences (cm)
No. of flower verticil/ principal inflorescence
No. of inflorescences/principal branch
Mean
s%
C=100
Mean
s%
C=100
Mean
s%
C=100
Control 
5 kR
15 kR
25 kR
3.3
3.3
2.6
1.4
58
60
63
32
100
102
78
27
4.5
4.4
3.7
2.1
42
46
46
38
100
97
82
46
35.4
35.1
27.8
11.3
62
61
64
43
100
99
79
32

In M1, the first vegetation year, the average height of the gamma irradiated plants, reduced progressively from 38.2 cm at 5 kR to 21.9 at 25 kR - a diminishing of 6-46% compared to the control; the number of branches/plant was drastically reduced at 25 kR (4.2 compared to the control: 32.6) and with 61-62% at 5 kR variants. The average length of the main inflorescence is slightly raised at 5 kR (33.4 cm to 32.6 cm) and, on the contrary, with 22% at 15 kR and 73% at 25 kR diminished. Compared to the smallest irradiation dose (5 kR), at 15 and especially 25 kR (compared to control) the flowering parameters reduced: the number of floral circles/main inflorescence (3.7-2.1; to control: 4.5) and the number of flowers in the inflorescence 35.1 to 11.3 (the control being 35.4), Tab. 1-2.

The results obtained in M1 (the firs vegetation year) we, generally, similar to those reported for other plant species that were the object of experimental mutagenesis with gamma rays.

Considering our aim, we may assent that gamma irradiation of hyssop could be a means to diversity the biological material. The proof is the fact that there are remarkably great differences among the irradiated variants and between this and the original populations. S% has often raised values of the plant height, 41-62 compared to 35 of the control, of 60 - 63 compared to in case of length of the inflorescence, 46 compared to 42 in case of the number of circles/inflorescence, 64 compared to 62 in case of the number of flowers/inflorescence (the case of the variant irradiated with 15 kR.

The 5 kR doses determined a stimulating effect in the second vegetation year, the plants being better developed than the control and other experimental variants.


Table 3. Variation of some morphological parameters of Hyssopus officinalis L. variants
treated with gamma rays, in M1 generation (2nd year of vegetation)
Variant
Plant height (cm)
Bush diameter (cm)
No. of branches/plant
No. of inflorescences/branch
Mean
s%
C=100
Mean
s%
C=100
Mean
s%
C=100
Mean
s%
C=100
Control 
5 kR
15 kR
25 kR
66.4
65.4
63.6
59.0
13
17
6
5
100
99
96
89
50.6
51.6
53.8
48.2
19
34
17
30
100
102
106
95
45.4
51.0
38.3
27.0
36
37
42
24
100
112
84
59
2.7
4.4
2.7
3.2
71
71
74
61
100
161
100
116

In the second year the length of the main inflorescence grow with greater irradiation doses 11.4 cm at 15 kR and 12.3 cm at 25 kR, compared to the control 10.6 (increases of 7% and 16% respectively) - Tab. 4; a similar effect was noticed in case of the parameter "number of floral circles/main inflorescence" which increased with 32% at the maximum irradiation dose.

On the contrary, the number of flowers/circle in the main inflorescence had reduced values compared to the control, the diminution being more and more significant with the growth of the irradiation dose.

The variability of the hyssop plants was also great in the second vegetation year of the mutagenized plants, s% having values of 30 - 34 in case of the bush diameter (the control: 19), 37-42 compared to 36 in case of the number of floriferous stems/plant, inflorescence length: 33 compared to 31, number of floral circles/inflorescence: 30-31 compared to 25 number of flowers/circle: 23-24 compared to 20 and number of flowers/inflorescence: 37-42 compared to 35.


Table 4.Some feature of Hyssopus officinalis L. inflorescences of gamma rays
treated plants, in M1 generation (2nd year of vegetation)
Variant
Principal inflorescences (cm)
No. of flower verticil/principal inflorescence
No. of flower/
verticil/principal inflorescence
No. of inflorescences/
principal branch
Mean
s%
C=100
Mean
s%
C=100
Mean
s%
C=100
Mean
s%
C=100
Control 
5 kR
15 kR
25 kR
10.6
9.8
11.4
12.3
31
29
33
33
100
92
107
116
12.3
11.1
12.3
16.1
25
30
31
30
100
91
101
132
6.7
6.9
6.6
5.1
20
24
23
24
100
103
98
77
82.6
77.4
76.1
64.5
35
42
37
38
100
94
92
78

In third vegetation year, the only variant exceeded in height the control was the one irradiated with 5 kR - 64.2 cm compared to 59.5 cm (Tab. 5). This dose had favourable effects on plant growth and development. Thus the bush diameter was greatest than that of the original plant - 58.8 compared to 47.3 cm (on increase of 29%); the number of floriferous stems (branching of the second order) increased with 26%; this variant had spike floriferouswith a better branching - 1.8 compared to 1.7 secondary spike floriferous(10% increase) - Tab. 5.


Table 5. Variation of some morphological parameters of Hyssopus officinalis L. variants
treated with gamma rays, in M1 generation (3nd year of vegetation)
Variant
Plant height (cm)
Bush diameter (cm)
No of branches/plant
No. of inflorescences/branch
Mean
s%
C=100
Mean
s%
C=100
Mean
s%
C=100
Mean
s%
C=100
Control
5 kR
15 kR
25 kR
59.5
64.2
54.4
51.5
10
7
11
8
100
108
91
41
47.3
56.8
43.5
42.2
27
22
18
11
100
129
92
89
148.2
187.1
110.5
95.7
38
46
35
26
100
126
75
65
1.7
1.8
1.2
1.3
77
98
49
68
100
110
71
77

At high irradiation dose, the length of the main inflorescence was not over that of the control (12 cm compared to 12.4 cm) but had more floral circles - 13 compared to 12 (increase of 9%), in their turn these more flowers - 9.6 compared to 9.3 (3% increase); the main inflorescence had more circles; the same variant had more flowers/main inflorescence - 124.3 compared to 113.5 (9-10% increase). In case of the other experimental variants, the characteristics of the inflorescence had values inferior to those of the control, decreasing with the increase of the irradiation dose. Thus (some exception at 15 kR) there were diminishing of up 59% at the inflorescence length, of up 24% in case of the floral circles and 14% in case of the number of flowers/main inflorescence (Tab. 6).


Table 6. Some feature of Hyssopus officinalis L. inflorescences of gamma rays
treated plants, in M1 generation (3rd year of vegetation)
Variant
Principal inflorescences (cm)
No. of flower verticil/principal inflorescence
No. of flower/ verticil/principal inflorescence
No. of inflorescences/ principal branch
Mean
s%
C=100
Mean
s%
C=100
Mean
%
C=100
Mean
s%
C=100
Control
5 kR
15 kR
25 kR
12.5
12.0
9.8
9.8
26
25
28
29
100
96
78
41
12.0
13.0
9.1
11.5
28
17
31
28
100
109
76
96
9.3
9.6
11.6
8.8
34
34
43
47
100
103
125
95
113.5
124.3
104.3
97.3
48
36
47
44
100
109
92
86

The data of Tab. 5 and 6 show the fact that in the 3rd vegetation year the effects of gamma rays on the growth and development of the Hyssop plants are also of inhibition, except only for the minimum dose (5 kR), which, as in the previous two years, stimulated investigated morphological characters.

Less variable was the bush diameter where s% had values of 11 at 25 kR to 22 at 5 kR (27 the control) and especially the plant height whom the variability coefficient oscillated between 7 (5 kR) to 11 (15 kR) (10 the control).

In yield of inflorescence, flowers, herb in general has a outstanding significance in the melioration process and consequently we shall tray - in our further experiment - to re-established the productive potential that was disturbed by gamma irradiation. If the biosynthesis of essential oils was stimulated - 0.93 g% at 5 kR and 15 kR, then 0.733 g% at 25 kR, compared to 0.362% at the control - the investigated morphological parameters were negatively affected (Tab. 4). In fact the level of essential oils was double that of the original population, the increases of this chemical parameter oscillating between 103% (25 kR) and 158% in case of the other two variants irradiated. In 1999 (the 3rd vegetation year) the content of essential oils in the variants treated with gamma rays is greater than in the original population: between 1.32 g% (25 kR) and 2.25 g% (15 kR) (Tab. 6). The relative values showed an increase of minimum of 60% (first variant) a maximum of 114% (the second).

The raised content of essential oils in the hyssop plants treated with gamma rays should be present in M2 too provided that this aspect be the result of favourable genetic events induced by gamma rays at the level of the genes implied in the biosynthesis of active principles. Franz, 1972, cited by Pop (11,13) concluded that heritability of the characteristic "content of essential oils" in hyssop (also of its components) might be high compared to other products of the plant secondary metabolites.

Further researches will, for sure, offer us new data to, in time establish the characteristic high essential oil content and to reefer to the utility of mutagen treatment to qualitatively and quantitatively embitter the drug of Hyssopus officinalis L. This aspect could encourage the melioration - via selection of this valuable medicinal and aromatic plant.


CONCLUSIONS

The treatment with gamma rays 5 - 25 kR induced a great variability, in all three-vegetation years. In the first generation (M1), the growth and developing process oh Hyssop plants were inhibited along with the increasing of the irradiation dose (from this point of view, the results are similar to those presented in literature).

The data obtained show that the agro-meteorolocical data in the Piatra Neamt area were favourable to the growth and development of Hyssop plants in 1998 (the morphological indices). For the content of essential oil, favourable was year 1999.

The 5 kR dose generally had stimulating effects, the plant height did not change (at greater doses there inhibitions of 10-20%), the bush diameter was with 11% greater than that of the control, probably also due to the greater number of stems - increase of 14%; the inflorescences had a better branching - 3.44 branches of the main spike floriferous (3 - the control, in increase of 15%). With the same dose (5 kR) we also noticed slight favourable effects at the level of number of flowers/plant (the number of main flower circles/main inflorescence grow with 2-3%, as well as the total number of flowers/inflorescence).

The treatment with gamma rays favoured the individual variability of Hyssop plants, which is anyhow rather great. S% (value of the variability coefficient) is often greater in the mutagenized plants than in the original De Ciorani population.

The analysis of the essential oil content in Hyssopi herba revealed a positive influence of the gamma rays treatment on the biosynthesis of the active principles: 0.733 g% at 25 kR to 0.934 g% at 5 kR compared to 0.362 g% (De Ciorani in the second vegetation year, 1998); 1.32 g% to 2.25 g% in the 3rd vegetation year (Tab. 7). Thus the meteorological conditions of 1999 were better than those of 1998 for the synthesis of essential oils.

We did not find a direct relation between the behaviour of the morphological indices and the essential oil level.


Table 7. Essential oil content in Hyssopus officinalis L. variants treated with
gamma rays, in M1 generation
Mutagen treatment
Essential oil (g%)
2nd year of vegetation
3nd year of vegetation
Mean
C=100
Mean
C=100
Control (C)
5 kR
15 kR
25 kR
0.362
0.934
0.930
0.733
100.00
258.10
258.01
203.14
1.051
1.703
2.250
1.322
100.00
161.90
214.29
125.71

LITERATURE
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[P-052]