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MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY 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 Biwen (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