Investigation of Flowering Dynamics of the Basil (Ocimum basilicum L ...

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Investigation of Flowering Dynamics of the Basil (Ocimum basilicum L.) and its Production Consequences. K. Szabó and J. Bernáth. Szent István University.
Investigation of Flowering Dynamics of the Basil (Ocimum basilicum L.) and its Production Consequences K. Szabó and J. Bernáth Szent István University Department of Medicinal and Aromatic Plants Budapest, Hungary, P.O. Box 53, H-1502 Keywords: flowering index, inflorescence, phenological phase, harvesting time, essential oil, GAP Abstract The flowering dynamics of Ocimum basilicum L. was studied with the aim to create an exact and practically applicable method for definition of its phases. Model experiments were done in 1997, on a common population maintained at the University and in 1998, the research work was extended over 11 populations of basil. According to our observation, the development of individual flowers of the basil can be characterised by 8 distinguishable phases, which must be considered for description of the actual phenological stage of a spike. An accurate model was created for the unambiguous description of flowering process of different flowers within the spike and for the individual plant as a whole. The new flowering index formula is calculated from the number of flowers weighted by their phenological phases. The time dependence of flowering is presented by functions fitted to values of the flowering index. The results reflected different patterns of the main inflorescence, the inflorescence formed on the side shoots in the first or in the second half of the flowering period. However, for description of the flowering process of the whole plant, a sigmoid function proved to be the appropriate model. The accumulation process of the essential oil could be characterised by the flowering index values (r=0.964). Based on the investigation of 11 genotypes it was proved, that the optimal harvesting time cannot be generalised to the species, as it is done by the literature. Genotype dependence was justified. INTRODUCTION Ocimum basilicum is an annual medicinal and aromatic plant from Lamiaceae family. Its main active agent is the essential oil, which accumulates during flowering in 0.5-1.5 per cent. In the course of research work with basil, the problem how to describe the phenological phases of sampling was raised, since the flowers within the basil’s spike open continuously parallel with the development of the spike. Looking through the related literature, numerous studies can be found trying to identify the phenological phase of sampling. Scientists can distinguish the phenophases according to the length of inflorescence (Bonnardeaux, 1992). Most frequently the estimation of the course of flowering is the way of the description (Basker and Putievsky, 1978; Lemberkovics et.al., 1993; Randhawa and Gill, 1995; Lemberkovics et.al., 1996; Gill and Randhawa, 1996; Gupta, 1996). Plugaru (1996) suggested deciding the harvesting time according to the maturation degree of the central spike; and in a study of Putievsky (1993) according to the maturation degree of the whole plant was described the phenophases of sampling. The description of phenophases varies from publication to publication. Therefore, the lack of exact definition of phenological phases decreases the reproducibility of samplings, the possibility of comparison of investigations and it produces in some cases contradictory results. In the present study, our intention was to characterise and describe the flowering dynamics of basil, giving numerical definition of the flowering stages. Furthermore, based on the proper numerical definition of the stages, a quasi model on development and essential oil accumulation was built-up. Proc. Int. Conf. on MAP Eds. J. Bernáth et al. Acta Hort. 576, ISHS 2002

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MATERIALS AND METHODS The open-field experiment was done in Soroksár, at the Experimental Station of the Department of Medicinal and Aromatic Plants, Szent István University, which can be described by continental climate and slightly humic sandy soil. During the growing seasons the plants were irrigated regularly. In 1997, the population of Ocimum basilicum ‘A-1’ was used for model experiments and in 1998, the research work was extended over 11 populations of basil. All the populations can be characterised by adequate morphological and chemical homogeneity. The plant material was grown by sowing seeds in greenhouse (April), thinning after 3 weeks and transplanting at the end of May. Each population consisted of 60 individual plants in spacing 60x40 cm. Two characteristics of populations were followed both years, the flowering and the essential oil accumulation. The study in floral biology was done from the beginning to the end of flowering period. Measurements were made every 3-7 days depending on the intensity of blooming, on three representative individuals of the populations. Developmental phases of flowers were registered within the inflorescence, continuously. For the proper identification, the apical inflorescence and those ones formed on the sideshoots were marked individually and ranked according to their position (Szabó, 2001). For checking the essential oil accumulation during the development, plants were harvested regularly. The samples taken from 3 average individuals each time, were dried and crushed. The distillation of essential oil was made by Clevenger apparatus in the laboratory of the Department using 10 g drug and expressed in % of the dry mass. For the mathematical evaluation SPSS statistical program was used. For modelling the time dependence of flowering, fitting function was applied. To describe the correlation between the essential oil content and the numerically defined phenological stages, correlation analysis was used. RESULTS AND DISCUSSION In order to characterise the ageing of inflorescence of basil the distinguishable phases of individual flowers was observed. According to our observation for flowers of basil 8 distinguishable phases exist: -green bud stage: the whorl has already separated on the axis of inflorescence, the petals are greenish, and from side-view the calyx still covers the corolla, -white bud stage: the petals are white and turgid, from side-view the calyx does not cover them, -opening of the flower: the petals are just before opening, or the stigma and stamina are already visible, however, the corolla is still tubulous, -fully open flower: the typical Labiatae corolla appears, the upper and lower lips diverge from one another, stigma and stamina show their characteristic colour, -fading of the flower: the stigma and stamen become brown, the petals show some kind of wilting, -white seed stage: from the abscission of petals to the development of full size of schizocarps -brown seed stage: the colours of fruits show the different shade of brown, their ripening began -black, ripe seed stage: characteristic colour and size of the schizocarps appear. In the model experiments in 1997 having registered all the counted numbers of flowers in the 8 stages, it was necessary to convert the mass of data into one number that facilitates to reach further conclusions. Therefore, based on literature data (Máthé, 1977) a flowering index formula to determine the developmental stage of the individual spikes or the plant as a whole was created. The simultaneous changes of different flowers within the spike were expressed by giving weights to developmental stages. Table 1. shows the grouping of stages and their weights. Thus the flowering index formula created for the exact determination of the developmental stage of basil inflorescence is as follows:

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∑(number of flowers in stages E…H X code of E…H) –∑(number of buds in stages A,B X code of A,B) ∑( number of flowers in stages A…H X code of A…H)

The value of flowering index calculated by this formula varies between number –1 and +1, theoretically. At the presence of budding stages only, its value is –1, and increases continuously afterwards by the appearance of more advanced stages. The flowering index value calculated by the codes of all flowering stages facilitates an unambiguous definition of the spike. These flowering index values could be calculated for the main inflorescence, its branches, and for each side-shoot. The results of one individual in 1997 are shown in Table 2. It can be observed that the index value of -1 lasts for several days, generally 2-5 days. The value never reached +1 because of the slowing down of plant development in autumn and its stop by the first frosts. The time dependence of flowering is presented by functions fitted to the flowering index values. The results reflected two different behaviours. Sigmoid function could be fitted to the values calculated for the main inflorescence and inflorescence formed on the side shoots which started their flowering in the first half of the flowering period (until 2530 of July). In contrast, the equations calculated for the flowering index of side-shoots appeared in the second half of generative phase, can be characterised by an irregular course. Each of the linear, quadratic, or cubic curves can be fitted to them only with low probability (r< 0.6). In a few cases within a short period (approx. 20 days) the curves show a characteristic and abrupt drop. It can be explained by the sudden opening of several flowers on the same inflorescence, causing a decrease of the flowering index. The changes going on the inflorescence developed in the second half of the generative period do not affect characteristics of the whole plant. These changes, considered irregular for side-shoots, are in balance and self-regulated within the whole plant. The irregular rhythm can be explained by the competition among seeds and flowers and among inflorescence with different ranks. Taking into consideration all flowering index values of every inflorescence formed on a single plant (main inflorescence and inflorescence formed on side-shoots of different ranks) sigmoid function could be fitted to the joint index values of any individual of Ocimum basilicum ‘A-1’(Fig. 1). The equation calculated is the following: Y= P1+(P2-P1)/(1+e-P3*(X-P4) ) P1 is the minimum value of the equation (= –1); P2 is the maximum point, equal to +1; P3 is the slope of the curve, which takes a value of about 0.2. P4 is the inflexion point referring to the time when the dynamics of flowering is slowing down. This point occurs on the 20-27th day counted from the beginning of the flowering. The curves describing the behaviour of the whole plant and that of side-shoots appearing in the first half of the flowering period in majority of cases are equal at a probability of 95 %. Thus, according to the statistical parameters of regression analysis, the curves of flowering index of side-shoots developing in the first half of the generative period, represent the flowering index of the whole plant properly. The described method of developing the formula of flowering index allows expressing the flowering stage of basil individuals in any actual moment, properly. On the next figure (Fig. 2), the courses of flowering index values of the 11 populations are shown. In most cases, the sigmoid curves appeared. The later the beginning of flowering of the population, the less the slope of the curve. Statistically proved the fitting of sigmoid curve in case of 6 populations. According to our results, the flowering index could be utilised also to define the phase when the essential oil attains its maximum level. Comparison of the essential oil accumulation and the flowering index data in a common system of co-ordinates is presented (Fig. 3). In the case of the studied population (‘A-1’ in 1997), the essential oil reaches its maximum value at the end of flowering period which is characterised by 0.8-

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0.9 values of the flowering index. The two parameters show close correlation (r=0,964). In case of population ‘A-1’ in 1998 (Fig. 4), results were similar to the previous year with the essential oil reaching a maximum value at the end of flowering, at flowering index value equal to 0.9. The same tendency was characteristic for the genotype called ‘Genoveser’ as well. In case of population ‘Rit-Sat’ (Fig. 5), the highest essential oil level was shown at about full flowering, at flowering index value equal to 0. The same accumulation rhythm was experienced at populations ‘Opal’, and ‘Keskenylevelű’ as well. The next figure (Fig. 6.) shows the results of population ‘Piroslevelű.’ This population, and populations ’OBRS’, ’Fodros’, ’Német’ produced the highest amount of essential oil at fading, at flowering index value equal to 0.5. The population ’Lengyel’ (Fig. 7.) produced the maximum of essential oil at early flowering, at flowering index value equal to –0.56. The same accumulation rhythm was experienced at population called ’USA’ at flowering index value equal to –0.74. CONCLUSIONS The definition of any phenological phase by the use of flowering index developed offers a new and objective method compared to those found in the earlier publications. It can be used also for the determination of optimal harvesting time. By these results it was shown, that "full-flowering" as generally accepted definition of the phenological phase when the highest essential oil level and yield can be obtained, may be inadequate. Based on the investigation of 11 populations genotype dependence of essential oil accumulation was justified. The application of this new method seems to be optimal in production of high quality drugs according to the requirements of the Good Agricultural Practice (Guidelines for GAP, 1998). ACKNOWLEDGEMENTS The research was supported by the OTKA found Nr. T020039. Literature Cited Basker, D. and Putievsky, E. 1978. Seasonal variation in the yields of herb and essential oil in some Labiatae species. J. of Hort. Science 53 (3):179-183. Bonnardeaux, J. 1992. The Effect of Different Harvesting Methods on the Yield and Quality of Basil Oil in the Ord River Irrigation Area. J. of Ess. Oil Res. 4. Jan - Feb. 65-69. Gill, B.S. and Randhawa, G.S. 1996. Effect of different transplanting dates and harvesting stages on the quality of French basil oil. J. of Herbs, Spices and Medicinal Plants 4(3):35-42. Guidelines for Good Agricultural Practice (GAP) of Medicinal and Aromatic Plants EUROPAM, 1998. Z. Arznei- und Gewürzpflanzen 3:166-178. Gupta, S.C. 1996. Variation in herbage yield, oil yield and major component of various Ocimum species/varieties (chemotypes) harvested at different stages of maturity. J. of Ess. Oil Res. 8:275-279. Lemberkovics, É., Nguyen H., Tarr, K., Máthé, I., Petri, G. and Vitányi, Gy. 1993. Formation of biologically active substances of Ocimum basilicum L. during the vegetation period. Acta Hort. 344:334-346. Lemberkovics, É., Petri, G., Nguyen, H., and Máthé, I. 1996. Relationships between essential oil and flavonoid biosynthesis in sweet basil. Acta Hort. 426:647-655. Máthé, Á. 1977. Az Adonis vernalis L. virágzásának számszerű kifejezése Herba Hungarica 16(2):35-43. Plugaru, V. 1996. Rolul si importanta inflorescentelor in lucrarile de ameliorare la busuioc (Ocimum basilicum L. ). Herba Romanica 13:35-47. Putievsky, E. 1993. Seed quality and quantity in sweet basil as affected by position and maturity. J. of Herbs, Spices and Medicinal Plants 2(1):15-20. Randhawa, G.S., and Gill, B.S. 1995. Transplanting Dates, Harvesting Stage and Yields of French Basil. J. of Herbs, Spices and Medicinal Plants 3(1):45-56.

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Szabó, K. 2001. A kerti bazsalikom (Ocimum basilicum L.) és a szurokfű (Origanum vulgare L. subsp. hirtum (Link) Ietswaart) kémiai, morfológiai és produkcióbiológiai differenciáltságának feltárása. PhD Dissertation, Szent István University, Budapest. Tables Table 1. The weights of the developmental stages in the flowering index formula Description of developmental stage A: green bud stage (bud stage) B: white bud stage (bud stage) C: opening of flower D: fully open flower E: fading of the flower (fading stage) F: white seed stage (fading stage) G: brown seed stage (fading stage) H: black, ripe seed stage (fading stage)

Weight of the stage in the formula 1 2 3 4 5 6 7 8

Table 2. Average flowering index values of main inflorescence, e1 e2 inflorescence and side shoots, I. individual (Soroksár, 1997). date main infl. E1 E2 17/7 -1 21/7 -1 24/7 -0,91 25/7 -0,53 28/7 -0,27 -1 -1 29/7 -0,08 -1 -1 30/7 0,107 -1 -1 31/7 0,179 -1 -1 1/8 0,207 -1 -1 4/8 0,494 -0,854-0,727 5/8 0,627 -0,765-0,563 7/8 0,658 -0,396-0,101 8/8 0,669 -0,173 0,173 11/8 0,652 0,237 0,178 12/8 0,661 0,287 0,237 21/8 0,765 0,765 0,736 25/8 0,846 0,878 0,845 5/9 0,964 0,947 0,948

K11 K12 K21

K22 K31

K32 K41 K42 K51

K52 K61 K62

-1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -0,914 0 -0,893 -1 -1 -1 -1-0,759 -1 -0,549 0 -0,743 -1 -1 -1 -1-0,568 0,135-0,094 -0,294 0,031 -0,512 -0,02 -0,864 -0,424 -0,787 0,18-0,008 -0,048 0,132 -0,473 0,059 -0,483 0,084 -0,455 0,226 0,222 0,303 0,187 -0,354 0,274 -0,206 0,048 -0,148 0,319 0,297 0,38 0,21 -0,255 0,39 -0,169 0,101 -0,062 0,701 0,626 0,459 0,585 0,424 0,496 0,569 0,406 0,687 0,821 0,652 0,683 0,641 0,569 0,648 0,561 0,625 0,715 0,97 0,892 0,827 0,879 0,848 0,839 0,856 0,815 0,856

-1 -1 -0,667 -0,704 -0,677 -0,42 -0,327 -0,157 0,442 0,244 0,744 0,671 0,223 0,595 0,87 0,765 0,782

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Figures

1 ,0

F lo w e rin g in d e

,5 0 ,0 -,5 P re dic ted va lue s

-1 ,0

O cim u m ba silic u m o bserve d valu e s

-1 ,5 0

10

20

30

40

50

D ay

Fig. 1. Mathematical modelling of flowering index values by sigmoid curve fitting.

flowering index

1 .5 1 0 .5 31Aug

25Aug

17Aug

10Aug

31-Jul

27-Jul

21-Jul

15-Jul

-0 .5

10-Jul

0

-1 -1 .5

day Fodros

O BRS

USA

Rit-Sat

K eskenyl.

O pal

Pirosl.

Genov.

N émet

A1

Fig. 2. Courses of flowering index values of the populations (1998).

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Lengyel

m l / 100g

index

1.2

1 0.8 0.6 0.4 0.2 0 -0.2 -0.4 -0.6 -0.8 -1

1 0.8 0.6 0.4 0.2

flow ering index

4-Sep

28-Aug

21-Aug

14-Aug

7-Aug

31-Jul

24-Jul

17-Jul

0

essential oil

Fig. 3. The courses of essential oil and flowering index values of ‘A-1’ through the growing season (1997).

m l/100g

index

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

1 0.5 0

essential oil

-0.5

f low ering index

25-Aug

17-Aug

10-Aug

31-Jul

27-Jul

21-Jul

-1

Fig. 4. The courses of essential oil and flowering index values of ‘A-1’ (1998).

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ml/1100g

index

1.2

1

1 0.5

0.8 0.6

0

0.4 -0.5

0.2

flow ering index

10-Aug

31-Jul

27-Jul

21-Jul

15-Jul

-1 10-Jul

0

essential oil

Fig. 5. The courses of essential oil and flowering index values of ‘Rit-Sat’ (1998). ml/100g

index

0.8 0.7

1

0.6 0.5

0.5

0.4 0.3

0

0.2 0.1

-0.5

flow ering index

17-Aug

10-Aug

31-Jul

27-Jul

21-Jul

-1 15-Jul

0

essential oil

Fig. 6. The courses of essential oil and flowering index values of ‘ Piroslevelű’ (1998).

ml/100g

index

1.4

1

1.2 1 0.8 0.6 0.4

0.5

essential oil

0

flow ering index

-0.5

0.2 17-Aug

10-Aug

31-Jul

27-Jul

-1 21-Jul

0

Fig. 7. The courses of essential oil and flowering index values of ‘Lengyel’ (1998). 112