Comparison Of Endogenous Hormones Levels Biology Essay

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Tree peony Paeonia suffruticosa a native to China, is a perennial ornamental plant of the genus Paeonia belonging to the family Paeonaceae. Species are divided in to tree and herbaceous peonies. The former is extensively cultivated in gardens for its beautiful flowers, pleasant fragrance and splendid color and also for cultural reason. Tree peony has long reputation in different countries and continents of the world. In China it is referred to as 'king of flowers'. Today, gardeners in Asia, North America, Europe and Australia regard tree peony as one of the most beautiful and most rewarding plant to grow.

The phenomenon of autumn-flowering is commonly observed in many horticultural plants, but for tree peony, this phenomenon is rare. Autumn-flowering tree peony is a cultivar with the habit of twice flowering in a year. In China, although there are abundant species and cultivars of tree peony, autumn-flowering cultivars are very rare. Therefore, in order to improve the resources of tree peony to meet up with its high market demand, much importance is needed to be attached to the collection, study and utilization of twice-flowering cultivars. The Japanese horticulturist have developed a cold-resistance tree peony called "Kan Botan" through making use of the habit of twice-flowering of some cultivars and many years of breeding

Autumn-flowering tree peony blooms twice in a year, in spring and autumn respectively. The flower buds develop normally in autumn and the florescence is mid or late October to mid November. Based on ancient literatures, autumn-flowering tree peony can bloom either in autumn or at the beginning of winter. Although autumn-flowering tree peony includes cultivars that bloom both in spring and autumn, the character of autumn flowering cannot remain stable, that is, not in all autumn that such cultivars develop terminal buds and bloom (Li, 2005).

Flower bud formation in plants is closely related to agricultural production and is the primary factor affecting yield and quality. Most perennials form flower buds and bear fruits mainly on spurs (Forshey and Efving, 1989). The presence of a sufficiently large area per spur is a significant factor favoring flower bud initiation (Huet, 1974). Like many other perennial woody plants, the flower buds of tree peony complete a change from vegetative to generative meristem in July or August each year after full bloom. Change from vegetative to flower buds is a complex process regulated by many factors, and can be influenced by endogenous hormones and assimilates, since they are known to affect differentiation and development of cells, tissues, and organs.

Nevertheless, the effect of these hormones varies even within the same plant species. For instance, a significant increase in the concentration of zeatin riboside (ZR) has been observed in normal chestnut tree that flowers and produces fruit only once in a year from June to July, whereas no increase is detected in the abnormal ones that flowers and produces fruit twice a year for the same period under the same condition (Liu et al., 2008). Besides, Bernier et al (1981) have suggested that the effect of ABA on flowering is diverse and species-dependent. Work by Liu et al (2008) fails to conclusively determine the role of ABA in chestnut flowering because the influence of ABA on flowering varies even within a single species.

Of recent time, cytokinin profiles in different plant organs have been reported to differ consistently with seasons. The level of cytokinin has been reported to be minimum in mid-June and maximum in late summer in apical buds of Abies nordmanniana. Sub-apical buds showed the same June minimum but peaked in mid autumn at a much lower level (Rasmussen et al., 2009). Pilate et al (1990) 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.

Hormones are known to 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, and flower development (Friml, 2003; Katia and Gilberto, 2004; Ana et al., 2004 cited by Liu et al., 2008). ABA has been reported to induce flowering (Shinozaki and Takimoto, 1983), promote flowering (Harada et al., 1971), and inhibit flowering (Nakayama and Hashimoto, 1973) in a short-day plant, P. nil. High level of IAA has also been suggested to promote flower bud development (Koshita et al., 1998) and produces more flowers in Satsuma mandarin (Citrus unshiu Marc.) (Koshita and Takahara, 2003) and decreases during flower buds initiation of tuberose (Ding et al., 1999).

GA3 is involved in several plant development processes and promote a number of desirable effects such as stem elongation, uniform flowering, reduced time of flowering, and increased flower size and number (Hopkins, 1995). GA3 has been reported to indirectly reduce flower bud formation in, avocado (Salazar-Garcia and lovatt, 1998), citrus (Lord and Eckard, 1978), apple and sweet chery (Koutinas et al., 2010) by counteracting the effect of auxin. Cytokinin stimulates cell division, shoot formation and help delay senescence or the aging of tissues. Reports have suggested that CTK controls the formation of flower buds in shoots (Koshita et al (1999) and determines the fate of buds (Rasmussen et al., 2009; Bollmark et al., 1995; Chen et al., 1996). According to Chen (1991), endogenous CTK levels in bud increase at the onset of floral initiation and differentiation in lychee.

Based on the above, it can be concluded that the literature on the role of hormones in floral induction, initiation and differentiation is vast and inconsistent. Therefore, the objective of this study was to determine the changes in endogenous hormones in leaf and node samples and assess their effects on flower bud formation during flower induction, initiation and differentiation cycle in autumn-flowering and non-autumn flowering of tree peony.

Materials and Method

Plant material

Ten field-grown plants of 5 years old autumn-flowering tree peony cultivar, each with at least 5 differentiated buds and of similar growth vigor, growing in Beijing will be chosen for investigation in summer (2010) in comparison to the same age of non autumn-flowering cultivar naturally grown in the field. The experiment was conducted at the Beijing Forestry University nursery, Jiu Fen.


Prior to sample collection, agronomic practices such as earthening, fertilization, pruning of feeble branches etc were carried out. A stainless clean knife was used to harvest samples and then rinsed with distill water. The collected bud samples were immediately placed in an ice box and upon taken to the laboratory, the samples were immediately dipped in liquid nitrogen (N2) and stored at -80OC until plant hormone extraction and analysis.

Sampling for plant hormone analysis

Hormone analysis was conducted during the current season growth on leaf and node. Samples were collected three times with a one-week interval targeting induction, initiation and differentiation periods. The sampling dates were 5th, 12th and 19th June for induction, 3rd, 10th and 17th July for initiation and 2nd, 9th and 16th August for differentiation.

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 ℃ 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, upon adding 1-2 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 ℃ 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.