Successive Secondary Flowering Mechanism In Tree Peony Biology Essay
Tree peony (Paeonia suffruticosa) is one of the most important ornamental plants grown in many parts of the world. The phenomenon of successive secondary flowering (autumn-flowering) is commonly observed in many horticultural plants including tree peony. Varying levels of hormones and sugar are thought to influence autumn-flowering. Therefore, the objective of this study was to evaluate the changes in the levels of endogenous hormones and sugars in the bud sample of 'Ao Shuang' cultivar of tree peony during the flowering period of autumn in comparison with that of spring. Hormones (IAA, ABA, GA3 and CK) and sugars (sucrose, glucose and fructose) levels were determined for both seasons.
The tree peony (Paeonia suffruticosa), native to China, and one of the most magnificent, most beautiful, and most interesting of all plants (Wister 1995), has come into its own as an important garden plant in many countries of Asia, America, Europe and Australia. It is an excellent ornamental plant that has been playing an important social, economic and medicinal role in the lives of the Chinese people. Respected as "the King of flowers" in China with a special cultural symbol of peace and happiness, prosperity and development, power and wealth over many other common plants, the tree peony has been extensively cultivated in China. Although having abundant species and cultivars of tree peony, China is rare in successive secondary flowering cultivars that flower twice or more in the same year. According to the history record and precedent researcher, China has autumn-flowering tree peonies resource, but however, the unstable bud sprouting and the lower flowering quality make them devaluing. If these rare resources are utilized by forcing them to re-flower in autumn will bring benefit to the Chinese, especially for the Nation Day celebration.
The development of flowering controlling mechanism in tree peony cultivation is very important because the plant growing in the field flowers only once a year with short flowering period. Due to the high demand and high price value associated with this plant, this phenomenon is undesirable for growers. In a production system of tree peony with at least 3-5 years before producing flowers, with a single bud for both leaves and flower production and where each planting is normally harvested only once, plant quality as well as flower quantity is of overriding importance for economic reasons and optimal conditions should be ensured in order to obtain maximum yield. This creates opportunities to probe into the flowering physiology of the plant under cultivation in order to enhance its production. Therefore, understanding the physiological mechanism of inducing successive secondary flowering in tree peony is indispensable in floriculture industry. Investigation of the relationship between secondary flowering and endogenous plant hormone levels is valuable to develop a technique/strategy for effectively controlling the quantity of flower during the year as well as to elucidate the physiological role of plant hormones in second flowering.
Several physiological changes occur in the shoot apex that specifically commits the apical meristem to produce flowers. The developmental changes that bring about flowering include endogenous factors, such as sugar changes, genes and hormones, and external factors, such as day length and temperature. Therefore, flowering, which represents the expression of reproductive phase, often depends on specific environmental and internal developmental signal. The transition from juvenile to adult phase often proceeds in response to environmental factors. These factors exert inductive effect by consequently causing changes in sugar and hormone levels (Zeevart, 1979). This phase change is a critical stage in plant reproductive development because it can be affected by changes in hormone and sugar levels consequently leading to seed and flower abortion and fruit abscission.
Tree peony naturally experiences a period of cool temperature before the buds grow out to produce shoots and flowers. Usually, flower bud initiation begins in the perennial 'crown' in later summer, as the leaves begin to senesce, then flower bud development continues till the plant enters dormancy (Byrne and Halevy, 1986). These buds will hardly develop further until a period of cool temperature has been experienced (Byrne and Halevy, 1986; Fulton et al., 2001; Halevy et al., 2002; Kamenetsky et al., 2003). As long as sufficient cool period of cool temperatures have been accumulated to break dormancy, then buds grow out to produce shoots and flowers during the warmer temperatures of spring (Kamenetsky et al., 2003). This phenomenon is different from peony cultivar such as 'Ao Shuang' which can also bloom in summer in the absence of cool temperature.
Peony, like many other flowering plants requires hormones for the growth and normal maintenance of their physiological and biochemical processes. As organic substances that regulate plant's growth, hormones play a central role in several plant developmental processes and promote a number of desirable effects including embryogenesis, lateral root development, vascular differentiation, apical dominance, climate responses, and flower development (Friml, 2003; Katia and Gilberto, 2004; Ana et al., 2004 cited by Liu et al., 2008). As a result, hormones are believed to strongly participate in flowering mechanism in most perennial plants including in tree peony.
Nevertheless, the effect of these hormones varies with environmental conditions at different period during the season (Koshita et al, 1999). For instance, according to Altman and Goren (1972), IAA delays summer bud sprouting, while GA enhances it and ABA completely inhibit it. Of recent time, cytokinin profiles in different plant organs have been reported to differ consistently with seasons. The level cytokinin has been reported to be minimum in mid-June and maximum in late summer in apical buds of Abies nordmanniana. Subapical buds showed the same June minimum but peaked in mid autumn at a much lower level (Rasmussen et al., 2009). Pilate et al., have also reported lower cytokinin concentration in the basal and median parts of conifer shoots than in the apical part at selected points in time. In addition, an inhibitory effect of exogenous IAA and ABA, and endogenous IAA were reported in Pharbitis nil by wijayanti et al (1997) in which exogenous GA induce flowering.
Moreover, high level of IAA was reported in branches that produced more flowers in Satsuma mandarin (Citrus unshiu Marc.) during the season when flower-buds developed (Koshita and Takahara, 2003). El-Antably et al (1967) indicated that ABA induce flowering in some short -day plants and inhibit flowering in some long-day plants. Lower level of zeatin riboside (ZR) and high GA levels has also been reported to influence flowering in abnormal tree of Chinese chestnut (Castanea mollissima) that flowers twice a year (Tao et al., 2008). Both levels of ZR and GA were observed at a period that coinciding the beginning of the second period of floral primordial formation in the abnormal tree. Besides, Nadia et al (2006) have reported that exogenous GA induces and promotes flowering in black iris (I. nigricans Dinsm.).
Contrarily, many previous studies have reported exogenous GA to inhibited flower bud formation not only in citrus (Monselise and Halevy, 1964; Goldschmidt and Monseile, 1972; Davenport, 1983; Koshita and Takahara, 1999), but also in pears (Griggs and Iwakiri, 1961), apples (Guttridge, 1962; Marcelle and Sironval, 1963), and cherries and peaches (Hull and Lewis, 1959). Koshita and Takahara (1999) reported that not only exogenous but also endogenous GAs reduced flower bud formation in citrus. Also works reported on other plant systems by Luckwill (1970), Su et al. (1979), and Koshita et al (1999) suggested that CTK controls the formation of flower buds in shoots. Moreover, work by Southwick and Davenport (1987) concluded that endogenous GAs and ABA might not play a major role in flower bud formation that was controlled by drought and chilling treatments. Thus, these results suggest the possibility that hormones are involved in flower bud formation and development but their influence is unclear and varies with plants.
Research on the relationship between hormonal changes and floral bud development may be of importance for the formulation of strategies and techniques for a year-round flowering programme in tree peony. In recent years, a year-round flowering programme has been reported by earlier and later forcing in Lilium rubellum through manipulating chilling temperatures, storage duration, and planting time (Ikeda, 1997). Lui (2003) found that few cultivars of tree peony could be forced for successive secondary flowering and suggested that the phenomenon is closely related to the number of flower buds differentiated and the type of differentiation gradient. Jiang et al. (2007) reported that tree peony cultivars such as Ru Hua Si Yu and High Noon with many flower buds and small differential gradient can be forced for successive secondary flowering. Li (1998) attributed this phenomenon to the wild species from which the cultivars were derived, while Xiao et al, (2001) attributed it to the endogenous phytohormone levels of the flower buds at different positions.
To find answer to these inconsistencies and understand the relationship between hormones and secondary flowering, this research was conducted to determining the dynamic changes of endogenous hormones and sugar levels in selected cultivars and assess their possible roles on flower bud development during successive secondary flowering in order to set up a protocol by which re-blooming ('out-of-season' technology) can be enhanced and flowering of tree peony could be promoted in tropical areas. Also, the information from this work could be useful for scheduling of the crop in flowering for specific marketing dates at different times of the year.
Materials and methods
The experiment was conducted at the Beijing Forestry University nursery, Jiu Fen during the summer of 2009 in comparison to the same cultivar naturally grown in the field in spring (2010). Eighteen plants of 5 years old tree peony, Ao Shuang cv. with similar growth vigor were chosen for investigation in August 2009 and February 2010 for the summer and spring season, respectively .
Prior to sample collection, agronomic practices such as earthening, fertilization, watering, pruning of feeble branches etc were carried out. Leaves defoliation was manually exercised in late August before dormancy-releasing treatment. The dormancy-releasing agent used was GA3 at 500 ppm applied at one dose on developed buds using a painting brush. A stainless clean knife was used to harvest sample buds from the plants and then rinsed with distill water. The collected bud samples were immediately placed in an ice box and upon taken to the laboratory, these samples were immediately dipped in liquid nitrogen (N2) and stored at -80OC until plant hormone extraction and analysis.
Sampling for plant hormone analysis
Bud samples were collected before and after defoliation and then after GA3 application. Sample collection after GA3 treatment was initially done at three days interval and later five days interval. The formal targeted bud developmental stages I, II, and III; stages at which abortion associated with flowering in tree peony is to a larger extent determined (Cheng et al., 2001), while the latter targeted bud developmental stages IV, V, VI and VII.
Hormonal extraction and analysis
The extraction of endogenous hormones was conducted as described by Chen (1991), with slight modifications. The content of endogenous levels of ABA, GA, IAA and CK was determined using fresh tissue (0.5g) of the bud. The plant tissue was grind with antioxidant (copper) and 10 ml of 80% cool methanol until it becomes homogenate and transferred into a test tube. Small amount of PVP was added into the homogenate, then spin the mixture on shaker for 10 min. and incubated at 4 â„ƒ overnight. The supernatant was transferred into 10ml tube the next morning and spin at 6000 rpm for 20 min. The residue was washed and re-extracted twice more with 2 ml of cold methanol for another 12 hours, and centrifuged under the same conditions as above and discarded the dust. The combine extracts, up on adding 2-3 drops of NH3, were evaporated (35-40OC) to the aqueous phase in a rotary evaporator. The aqueous phase was then dissolved by adding some distilled H2O followed by the separation of the mixture into two equal parts. One part of the mixture was adjusted to pH 2.5-3.0 with 1N HCl and then partitioned three times with equal volumes of ethyl acetate. The combine ethyl acetate fraction was evaporated to dryness. The dry part was diluted with 3% methanol and 97% 0.1 M HAc for the determination of acidic hormones such as IAA, GA3 and ABA. The second part of the mixture was adjusted to pH 7.5-8.0 with 1N NH3, and also partitioned three times with equal volume of pH 8.0 phosphate buffer saturated with n-butanol (butanol in phosphate buffer).The combined mixture was evaporated at 60 â„ƒ until no liquid left. The dry part was dilute with 3% ammonia and 97% pH 7.0 of pure water for the determination of CTK.
Determination of hormone
Hormonal determination was done according to the HPLC method, Agilent 1100 chromatography, C18 tube (250*4.6 mm), matrix contest. Mobile phase: 3% methanol and 97% 0.1 M HAc for IAA, GA3 and ABA; and 3% ammonia and 97% pH 7.0 pure water for CTK. Wavelength of contest of different hormones: IAA-280 nm, ABA-260 nm, GA3 -210 nm, CTK-267 nm, speed of flood is 1 ml/min, contest with "outer-standard method" and the standard sample used is production of Sigma.
The extraction of endogenous hormones was conducted as described by Zheng et al. (1999), with some modifications. The content of endogenous levels of ABA, GA, IAA and CK determined using fresh tissue (0.5g) of the bud. The plant tissue (freeze-dried powdered) was be placed, stirred and extracted in a 10ml chilled extraction medium containing 80% methanol and kept overnight at low temperature. The homogenate was centrifuged for 15 min. at 10,000 rpm, 4OC and the supernatant was collected and transferred to a flask. The residue was washed and re-extracted twice more with 2 ml of cold methanol for another 12 hours, and centrifuged under the same conditions as above. The supernatants were pooled, compressed and de-colored using Sep-Pak C-18 cartridge twice so that the dilution curve will be parallel to the curve of the standards. After filtration, the supernatants will be dried in vacuum at 37OC to remove menthanol. The residue will be re-dissolved in a buffer containing 0.05 mM 1 Tris, 1 mM MgCl2, 150 mM NaCl, and 0.1% Tween 20. The measurement of hormone contents will be conducted by GC-MS (Gas Chromatography-Mass spectrometry).
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