Summary of the Phd thesis dissertation in Biology: Studying the effect of some factors on the flowering process of Torenia fournieri L. cultured in vitro

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The objective of this project is determination the role of a number of factors such as LED light, type and concentration of sugar, plant growth regulators, mineral medium, amino acids and polyamine in the flowering of T. fournieri in vitro.

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Nội dung Text: Summary of the Phd thesis dissertation in Biology: Studying the effect of some factors on the flowering process of Torenia fournieri L. cultured in vitro

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ----------------------------- STUDYING THE EFFECT OF SOME FACTORS ON THE FLOWERING PROCESS OF Torenia fournieri L. CULTURED IN VITRO Area of specialization: Plant physiology Code: 9 42 01 12 THE ABSTRACT OF PHD DISSERTATION IN BIOLOGY HCM CITY – 2020
The dissertation was performed at Institute of Tropical Biology and Tay Nguyen Institute of Scientific Research, Vietnam Academy Science and Technology Supervisor: Prof. PhD. Duong Tan Nhut PhD. Nguyen Huu Ho Reviewer 1: Reviewer 2: Reviewer 3: The dissertation will be defended at the PhD dissertation committee of Graduate University of Science and Technology, Vietnam Academy Science and Technology at on , 2020 The dissertation was deposited at: - Library of Graduate University of Science and Technology - National Library of Vietnam
INTRODUCTION T. fournieri is beauty flower, easy care and can be grown for many different purposes such as potted, roadside, indoors, outdoors, grown in cashew, hot, moderate or cold climates. In addition to the ornamental value, T. fournieri is a useful experimental model plant for researchers, especially suitable for research of basic physiological phenomena because of its short life cycle, responding quickly to the change of culture medium. T. fournieri is also used as a source of materials for in vitro studies such as gene transfer, somatic mutation, micropropagation, in vitro flower production... Although there are some researches on flowering of T. fournieri in vitro, studying the role of some factors such as plant growth regulators, carbon sources, mineral nutrient media polyamine, amino acids in the flowering process is necessary to explain more clearly the flowering mechanism of this plant. Plant Cell tissue culture and molecular biology techniques have contributed to the knowledge of flowering processes in plants. So far, besides A. thaliana, a model plant that has been used extensively for flowering research, T. fournieri has also been used as a source of material for in vitro flowering study. In the study of flowering in plants, in vitro culture conditions are ideal for carrying out further studies of this process. Scientists can actively adjust a number of factors such as light intensity, light, temperature, sugar, minerals medium, plant growth regulators in laboratory condition... depending on research purposes. Changes in medium composition, plant growth regulators, or specific changes in culture conditions may accelerate growth rate, shorten the growth period in order to lead early flowering. With this technique, scientists are aiming to further investigate the physiological phenomena 1
of the flowering process. This is an important step on the road to conquer nature. Stemming from the above problems, the topic "Studying the effect of some factors on the flowering process of Torenia fournieri L. cultured in vitro" was carried out. The objective of the topic The objective of this project is determination the role of a number of factors such as LED light, type and concentration of sugar, plant growth regulators, mineral medium, amino acids and polyamine in the flowering of T. fournieri in vitro. Research content Content 1. Study effect of some factors on the flowering of T. fournieri cultured in vitro. Content 2: Observe changes in morphology, structure of meristem and metabolic activity in the process of flowering of T. fournieri cultured in vitro. CHAPTER 1. LITERATURE REVIEW 1.1. Introduction to the Torenia fournieri (T. fournieri) Classify v Kingdom: Plantae v Phylum: Magnoliophyta v Class: Magnoliopsida v Order: Lamiales v Family: Scrophulariaceae v Genus: Torenia v Species: Torenia fournieri L. The appearance of T. fournieri is described as follows: 2
Stems: green, 15-35 cm in height, often upright. Stems erect, quadrangular, simple or branched above middle, subglabrous Leaves: glossy green, up to 5 cm in length, simple, ovoid (oval), with serrated margin, oppositely arranged on the stem. Flowers: diverse in color and shape. The traditional colors include: purple, blue; the new colors are: white, red, pink, dual color, yellow. Flowers are borne on stalks (pedicels) and may be solitary or may be part of an inflorescence that is classed as a cyme or raceme. T. fournieri is a perennial, dicotyledonous herb, but it is often grown as annual plants. T. fournieri is a diploid plant (n=9). Tetraploid plants (n=18) can be produced by treating the colchicine of plantlet in order to produce commercial varieties with large flowers. However, these tetraploid plants, significantly reduced the viability of pollen, grain formation and uneven chromosome distribution at the anaphase stage when compared to plants bisexual parents. T. fournieri is the favourite flower because of its variety of colors, easy care and is suitable for many purposes such as potted, ornamental, fence or plant to cover the ground. In addition to the decorative value, T. fournieri is also a useful model plant for cytologists to study hybridization because it is easy to perform due to protruding of the embryo sac. T. fournieri is also used to study the position and movement of chromosomes and centromere at an early stage of embryogenesis in hybrid plants. 1.2. The path to flowering in plants The evolutionary mechanisms of plants take place strictly to ensure that flowering must occur at the optimal time for successful reproduction. These regulatory mechanisms require the combination of many environmental factors together with endogenous factors so that 3
the transition of flowering takes place at the right time. Flowering pathways in plants include photosynthesis, energy pathways, plant growth regulators and autonomous pathways were studied from Arabidopsis thaliana, model plant. However, not all plants flower in exactly these four basic ways. Depending on the species, each specific living condition, plants will have different stimulating factors suitable for flowering. 1.3. Domestic and foreign research achievements on in vitro flowering and T. fournieri 1.3.1. A number of domestic studies Plant growth regulators have always played an important role in plants and flowering control because these growth regulators have been studied quite extensively. Le Hong Thuy Tien (2006) studied in vitro flowering of Catharanthus roseus and Petunia hybrida, successful flowering when shoots were cultured on MS medium supplemented with 0.05 mg/l TDZ and 0, 1 mg/l NAA. The most used combination of cytokinin and auxin to induce flowering of Saintpaulia ionantha Wendl, Celosia cristala, or Rosa hybrida are BA and NAA (possibly IAA, IBA). In addition, GA3 has also been used for inducing in vitro flowering in Dischidia pectinoides. In addition to inducing flowering, plant growth regulators also play an important role in flower development or shorten flowering time. In the Celosia cristala, PBZ at 2.0 mg/l increased flowering rate and induced early flowering, the duration was 30 days instead of 50 days after culture. Kinetin at a concentration of 1.0 mg/l promotes the formation of flowers; meanwhile more new shoots were appeared at lower concentrations. For Helianthus annuus, flower was formed in medium supplemented with 50 g/l sucrose combined with 1.0 mg/l IBA. 4
1.3.2. Some studies abroad Currently, there have been many studies on in vitro flowering in Dendrobium orchid species such as D. madame, D. chao-praya-smile and D. candididum. For other orchids such as C. goeringii and C. hybridium, in vitro flowering was also recorded in the study of Li-Ming and Ji-Liang (2006). In vitro flowering induction was studied in Pharbitis nil, Passiflora suberosa and Lilium longiflorum by using cytokinin and gibberellin. In 2013, Zeng et al. successfully studied in vitro flowering in mini rose (Rosa hybrida cv. Fairy Dance). In vitro flowering has also been studied when culturing somatic embryos in some plants such as B. vulgaris and D. giganteus. John and Nadgauda (1997) studied in vitro flowers in the Bambusa vulgaris var. vittata and B. arundinacea. Some other bamboo species have been used as a source of materials for in vitro flowering research such as B. edulis, D. hookeri and D. latiflorus. In vitro flowering has been successfully studied for several medicinal plants. Seidlova et al. (1981) studied the effects of ABA on flowering of Chenopodium rubrum, short-day plant. For ginseng (Panax ginseng), Tang (2000) investigated in vitro flowering from embryo culture. Changes in endogenous growth regulators content in differentiation of flower buds and vegetative shoots in thin cell layer culture of Cichorium intybus were investigated by Ying-zhang and Bi- wen (1996). In vitro flowering has been studied in valuable medicinal plants such as Solanum nigrum, Centaurium pulchellum and Chamomilla recutita. Vadawale et al. (2006) successfully studied flowering in Vitex negundo cultured in vitro. 5
CHAPTER 2. MATERIALS AND METHODS 2.1. Material T. fournieri, a purple-white flower, is used as an initial source material. The leaves of T. fournieri ex vitro are pre-sanitized on surface with light soap and placed under running water for 30 minutes. The explants were steriled in 0.1% HgCl2 solution with 1-2 drops of Tween 20 for 5 minutes. The explant was then rinsed 3 times with sterilized distilled water and then cultured on Murashige and Skoog (MS) medium supplemented with 30 g/l sucrose, 8 g/l agar and 0.5 mg/l BA for shoots regeneration. After that, these shoots were cultured on MS medium in order to create a healthy and homogeneous shoots; shoots with height of 2.0 cm were used as the materials for experiments. 2.2. Experimental design 2.2.1. Content 1: Study the role of some factors on the flowering of T. fournieri in vitro 2.2.1.1. Experiment 1.1: Study the role of light on the flowering of T. fournieri plants cultured in vitro 2.2.1.2. Experiment 1.2: Study the role of sugar on the flowering of T. fournieri cultured in vitro 2.2.1.3. Experiment 1.3: Study the role of exogenous plant growth regulators on the flowering of T. fournieri cultured in vitro 2.2.1.4. Experiment 1.4: Study the role of the mineral medium on the flowering of T. fournieri cultured in vitro 2.2.1.5. Experiment 1.5: Study the role of amino acids (L-tyrosine, arginine and proline) on the flowering of T. fournieri cultured in vitro 2.2.1.6. Experiment 1.6: Study the role of polyamine (spermidine, spermine, and putrescine) on the flowering of T. fournieri cultured in vitro 6
2.2.2. Content 2: Changes in morphology, structure of apical meristem and metabolic activity during flowering of T. fournieri cultured in vitro 2.3. Culture condition The experiments were placed under lighting conditions with the photoperiod of 10 hours/day, light intensity of 45 ± 2 µmol m-2 s-1, temperature: 25 ± 2°C, humidity: 60 ± 5%. 2.4. Research methods • Plant cell tissue culture method • Anatomy of meristem morphology • Methods of measuring chlorophyll content in plants • Methods of measuring total sugar content in plants • Methods of measuring photosynthesis and respiration intensity in plants • Methods of measuring leaf area • Methods of data collection, statistics and processing 7
CHAPTER 3. RESULTS 3.1. Content 1. Effects of several factors on the flowering of T. fournieri in vitro 3.1.1. The role of lights on the flowering of T. fournieri in vitro One of the important pathways in flowering induction in plants is the light path. The quality, intensity and duration of light affect directly plant growth and development. The study of Duong Tan Nhut et al. (2013) on T. fournieri showed that T. fournieri culturing in lighting conditions with the intensity of 45 µmol m-2 s-1 and the lighting time is 10 hours/day, after 60 days, the plants had strong stems, green leaves and high flowering rate (70.06%). Inheriting previous studies, in this study, the quality or wavelength of light was investigated to determine the role of light wavelength on the flowering of T. fournieri cultured in vitro. Different light qualities influenced the flowering of T. fournieri cultured in vitro differently. After 40 days of culturing, the results in table 3.1 showed a statistically significant difference between treatments using LED and fluorescent lamps. The highest flowering rate of in vitro T. fournieri was 19.59% at 50% red LED combined with 50% blue LED with number of flower/shoot was 0.33. Increasing the rate of red LED light to 60%, the flowering rate decreased to 13.34%. But this rate started to decrease rapidly when the ratio of red LED light exceeds 60%. This is consistent with the work of Dewir et al. (2006) on Euphorbia millii, in vitro flowering decreased with increasing the ratio of red LED. 8
Table 3.1. Effect of light condition on the growth and development of T. fournieri in vitro after 40 days of culture. The flower No. of flower Shoot Leaf area Experiments formation rate (%) /explants height (cm) (cm2) d a HQ 0,00 0,00 6,60 1,33a Xanh 0,00d 0,00 5,00b 0,94bc Do 0,00d 0,00 5,90ab 0,72c a a 5Đ:5X 19,59 0,33 6,66 1,31a 6Đ:4X 13,34b 0,13 6,32a 0,90bc 7Đ:3X 0,59c 0,06 6,33a 0,79c 8Đ:2X 0,61c 0,06 5,77ab 0,76c d ab 9Đ:1X 0,00 0,00 5,84 0,69c P * ns * * In the same column, the numbers with different followed letters are statistically different at 5%. (*); ns: no significant; Do: 100% red LED; Xanh: 100% blue LED; 9Đ:1X: 90% red LED + 10% blue LED; 8Đ:2X: 80% red LED + 20% blue LED; 7Đ:3X: 70% red LED + 30% blue LED; 5Đ:5X: 50% red LED + 50% blue LED; HQ: fluorescent light (Philip, 36W, 1,2 m). Research results of Heo et al. (2006) also showed that the rate of the flowering along with the number of flower of vines was higher when using red LED in combination with green LED and much higher than using LEDs only in a single wavelength type (blue or red). The results of in vitro studies on T. fournieri are similar to those of Heo et al. (2006). There was nearly no flowering induction when placing buds under lighting conditions of 100% red LED or 100% blue LED. The flowering induction of T. fournieri only occurred by the appropriate signal of the blue LED light in combination with the red LED light. This can be explained that red LED light stimulates phytochrome activity, blue LED light stimulates cryptochrome operation; The combination of blue LED and red LED light creates an appropriate ratio to stimulate the expression of genes involved in flowering induction such as the flc gene, one of the genes encoding the MADs-box protein. From there, flc activity promotes later flowering sequences including stimulation of the activation of a combination of genes that determine flowering time such 9
as ft, soc1 and lfy, and stimulates activity of meristem, eventually leading to flowering. 3.1.2. The roles of types and concentrations of carbohydrate on the flowering of in vitro T. fournieri The results of carbohydrates experiments on the in vitro flowering rate showed that the highest flowering rate of T. fournieri was 53.14% when shoots were cultured on MS medium supplemented with 50 g/l fructose, followed by glucose at a concentration of 50 g/l (51.48%) (Figure 3.1). However, in the fructose experiment, in vitro plants became yellow, browned and died, the percentage of dead plant explants was relatively higher than the experiment on other sugars. Therefore, 50 g/l glucose was the most suitable for the growth and development, especially for the flowering induction of in vitro T. fournieri. Studied on in vitro T. fournieri, two types of monosaccharides (fructose and glucose) affect the in vitro flowering more efficiently than disaccharides, such as sucorse and maltose. The results of this study are similar to those of Taylor et al. (2007). In their research in 2007, the results demonstrated that the flowering rate of K. leucocephala increased when the shoots were cultured on medium supplemented with monosaccharides, which is explained by the effect of this saccharide on cell division in apical meristem tissue, this is important for the flowering induction in plants. Glucose and fructose do not always stimulate the induction of flower formation, so this effect may be uncommon and may depend on plant species, the nature of the explants and the ability of the explants to use different carbon sources. 10
Figure 3.1. Effect of type and concentration of sugar on flowering of in vitro T. fournieri after 40 days of culture. The distinction between inductive flowering and flower development in relation to sugar activity is an issue to consider. While a particular sugar may not stimulate flower induction, it can have a positive effect on flower development as noted in T. fournieri. In this study, although sucrose did not significantly affect the induction of flower formation, in the previous study on T. fournieri, when adding sucrose at a concentration of 60 g/l to the induction medium, flower (¼MS medium), the rate of flowering as well as the number of flower buds is higher than the control treatments (treatment using induction 11
medium). This also showed that sucrose does not play a role in induction of flowers but only promotes the development of T. fournieri in vitro. 3.1.3. The roles of plant growth regulators on the flowering of in vitro T. fournieri Effects of GA3 in combination with ABA on the flowering induction of T. fournieri cultured in vitro Tanimoto et al. (1985) showed that flower formation of T. fournieri was influenced by endogenous factors inside the explants, including the content of endogenous ABA. Therefore, the addition of 0.1 mg/l ABA to the culture medium stimulated flower formation of in vitro T. fournieri shoots. This result is consistent with the results of this experiment, when using ABA at different concentrations induced flower formation. In medium supplemented with 1.5 mg/l ABA, the rate of flower formation was higher than other treatments and control treatment. The flower formation rate of that treatment was 80% higher compared to 40% of control treatment (Table 3.2). This suggests that ABA plays an important role in the induction of flower formation of T. fournieri. A number of recent studies showed that the effect of ABA on flowering induction can be explained by the expression of the genes abi3, abi5 and basic leucine zipper, which play an important role in sensing flowering by regulating flc gene expression. Another protein that is also involved in the control of flowering is HAB1 (HYPERSENSITIVE TO ABA1), which is the inhibiting factor of the response of ABA signals. Expression of the HAB1 protein was found while inducing the flower of A. thaliana. The HAB1 protein interacted with the SWI/SNF protein complex. The authors suggest that the 12
interaction of HAB1 protein and SWI/SNF complex (chromatin complex) in transcription in response to necessary ABA signals for the regulation of genes involved in ABA signal response. There are many miRNA genes that have also been associated with ABA flowering. The gene miR159 involved in flower development, which controlled the expression of myb101, myb33 is also regulated by ABA. The miR160 gene that controlled flower morphogenesis was also controlled by ABA. On the other hand, overexpression of miR172c reduced plant water loss and promotes early flowering by controlling the expression of ft and lfy genes under long day conditions. The miR172 gene was induced by ABA, which was related to drought, which increased sensitivity to ABA, accelerating response to drought. In this study, in experiments surveyed the effect of individual GA3 or GA3 in combination with ABA at different concentrations on the flowering of in vitro T. fournieri, after 40 days of culture, the results were obtained. It showed that the flowering rate in these treatments was lower than the treatments using ABA individually and control treatment. This suggests that GA3 had a negligible influence on the flowering induction in this plant. The role of GA3 in the flowering induction in plants that were not dependent on photoperiods has not been specifically proven, probably that GA3 is a factor that promotes flowering in this plant but does not effect on the flowering in another species. 13
Table 3.2. Effect of GA3 in combination with ABA flowering of T. fournieri in vitro after 40 days of culture PGRs The flower No. of flower Shoot GA3 ABA formation rate (%) /explants height (cm) (mg/l) (mg/l) 0,0 40,00bc 5,92bcd 0,63bc 0,5 40,00bc 6,37b 0,63bc 0,0 1,0 46,67ab 5,58bcd 0,73ab 1,5 80,00a 8,08a 1,33a 2,0 20,00bcd 5,45bcd 0,30bc 0,0 13,33bcd 5,60bcd 0,20bc 0,5 26,67bcd 5,08bcd 0,37bc 0,5 1,0 20,00bcd 6,41b 0,27bc 1,5 23,33bcd 6,19bc 0,47bc 2,0 0,00d 5,43bcd 0,00c 0,0 0,00d 6,12bcd 0,00c 0,5 0,00d 6,37b 0,00c 1,0 1,0 0,00d 5,56bcd 0,00c 1,5 3,33d 5,73bcd 0,07bc 2,0 10,00cd 5,98bcd 0,13bc 0,0 6,67cd 5,91bcd 0,10bc 0,5 6,67cd 5,27bcd 0,13bc 1,5 1,0 0,00d 4,63cd 0,00c 1,5 3,33d 4,59d 0,03c 2,0 10,00cd 4,65cd 0,17bc 0,0 3,33cd 5,32bcd 0,03c 0,5 10,00cd 5,59bcd 0,10bc 2,0 1,0 0,00d 5,50bcd 0,00c 1,5 0,00d 5,43bcd 0,00c 2,0 3,33d 5,11bcd 0,03c PGA * * * PABA * * * PGA*ABA * * * In the same column, the numbers with different followed letters are statistically different at 5%. (*). 14
Effects of GA3 in combination with BA on the flowering induction of T. fournieri cultured in vitro The results showed that in medium supplemented with BA in combination with GA3, in vitro T. fournieri, there was no flower formation, only new shoots formation, and an average height of shoots is 3.0 cm. The plants are high, vitrification and weak. Similar to the results on the study of Tylophora indica, GA3 was reported to be suitable for in vitro shoot regeneration, growth promotion, biomass production and xylem prolongation. Effects of BA in combination with NAA on the flowering induction of T. fournieri cultured in vitro Similar to treatment GA3 in combination with BA, after 40 days of culture on MS medium supplemented with BA and NAA, the results also showed that these two types of plant growth regulators did not affect the flowering of in vitro T. fournieri. On medium supplemented low concentration of BA (0.5 mg/l BA), the average height of shoots was higher than explants on medium with high concentration BA and root formation was observed in explants. 3.1.4. The roles of mineral medium in the flowering of T. fournieri cultured in vitro In order to evaluate the effects of the mineral medium on the growth and flower formation of in vitro T. fournieri, the experiment was conducted with MS medium and its modifications. After 40 days of culture, the rate of flower formation on the ¼MS medium was 60.00%, which was higher than the control and the other media. The macromolecular components in ¼MS and ½MS medium were reduced in proportion to ½, ¼ and the micromolecular contents was constant compared to MS medium. These modifications led to a change 15
in C/N ratio in the medium. This can be explained that high concentrations of macronutrients, especially N, presented in medium would partially inhibit the transition from vegetative to reproductive phases or the flower bud formation of T. fournieri. Reducing the amount of macro minerals led to a decrease in N content, but keeping C content, led to a high ratio of C/N, which stimulates flowering. Conversely, in a low C/N ratio medium, nutrient generation prevailed. According to Tanimoto and Harada (1981), the C/N ratio played an important role in the transition of the flowering in plants. The ratio of C/N in the ¼MS medium is higher than in MS medium, which made explants go into reproductive state, this result was also consistent with the study of Wang et al. (2002). Table 3.3. Effect of mineral medium on the flowering of T. fournieri in vitro after 40 days of culture The flower No. of Shoot height Experiment formation rate (%) flower/explants (cm) ½MS 43,33ab 0,47 6,20b MS½ 23,33bc 0,33 6,34b ¼MS 60,0a 0,70 8,70a MS¼ 6,67c 0,07 9,13a MS 16,67bc 0,27 5,25b P * ns * 3.1.5. The roles of amino acids (proline, arginine and L-tyrosine) in the flowering of T. fournieri cultured in vitro The results showed that flower formation varied depending on the types and concentrations of amino acid, which were added to culture medium. For T. fournieri, 1.5 mg/l of arginine is the most suitable for flower formation (Figure 3.2). The roles of amino acids on flowering induction were still unclear, because in most treatments using amino 16
acids, the flowering induction only appears after 80 days of culture, compared to other factors, the flowering occurred on day 40th. Figure 3.2. Effects of amino acids on T. fournieri in vitro flowering after 80 days of culture. P1.0, P1.5, P2.0, P2.5, P3.0, P3.5, P4.0, P4.5, P5.0: corresponds to proline in concentrations from 1.0 to 5.0 mg/l; A1.0, A1.5, A2.0, A2.5, A3.0, A3.5, A4.0, A4.5, A5.0: correspond to arginine in concentrations from 1.0 to 5.0 mg/l; T1.0, T1.5, T2.0, T2.5, T3.0, T3.5, T4.0, T4.5, T5.0: correspond to L-tyrosine in concentrations from 1.0 to 5.0 mg/l. 17
3.1.6. The roles of polyamines (spermidine, spermine, putrescine) to the flowering of T. fournieri cultured in vitro Polyamines (PAs) are considered as a group of plant growth regulators; therefore, the effect of PA on the flowering induction depends on plant species, concentration and types of PA. Five important biosynthetic enzymes related to PA biosynthesis in higher plants are currently being studied: arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC), Spd synthase (SPDC) and Spm synthase (SPMS). The important physiological functions of endogenous PA in flower induction, organ and preliminary flower formation have been confirmed in various studies. Triamine Spd, the most important of the three PAs mentioned above, is involved in many physiological processes. In Polianthes tuberosa, Spd and Cad are the markers for flower differentiation. Experiments on Cucurbita pepo and Ginkgo biloba also found that Spd dominating the flowering differentiation. When the Spd reaches a certain level will lead to the promotion of the expression of flowering genes; thereby, activating the synthesis of a particular protein and finally the preliminary flower formation. This also explained the important role of exogenous Spd in the flowering of in vitro T. fournieri. In this study, different types of polyamine effect on different flowering induction of in vitro T. fournieri. Spermidine and spermine were suitable for the flowering induction of T. fournieri. The flowering formation rate reached 43.33% and the number of flower /explant reached 0.47 flower buds when culured the T. fournieri explants on medium supplemented with 40 mg/l spermidine. The flower formation rate of T. fournieri culturing on medium supplemented with 40 mg/l 18