| The growth rhythm of Brassolaeliocattleya Sung Ya Green'Green World'was observed and recorded in greenhouse in North China, and also the temperature and relative humidity was recorded. The changes of nutrient content in organs of different physiological ages and the chlorophyⅡfluorescence characteristics in leaves of different age were studied. The flower bud differentiation process was observed by the method of paraffin cut. The changes in nutrient content and endogenous hormones in new leaves were measured during the flower-bud differentiation phase of plants growing under 3 different temperatures. The effects of different temperature treatments, spraying or injecting different concentration of hormones (GA3,NAA,ABA) on Cattleya florescence and flower quality were studied. Physiological and biochemical changes in flower senescence were also studied. An antisense expression vector of the cattleya-ACO gene, which named pBI121ACC, was constructed. This provides the foundations of future transgenic research for prolonging the flowering period of transgenic Cattleya via antisense technology. The results are described as follows:Soluble sugar was the dominant carbon reserves with lower content of starch in new pseudobulbs, but starch was dominnant in older (2-4 years) pseudobulbs and leaves. Most of carbon nutrient was consumped during flower bud differentiation and florescence. There was lower nutrient storing during dormant period, but more during new bud sprouting. Therefore, new bud sprouting period appears to be the best time for repotting and division propagation.With increasing of light intensity and leaves age, there was a slower decrease in actual PSII efficiency(Yield), photochemical quenching (qP), and efficiency of energy conversion of open PSII (Fv'/Fm'), but a slower increase in nor-photochemical quenching (qN). The new leaves under lower light intensity had higher qP, Yield, Fv'/Fm', but it is susceptible to high light intensity under which photoinhibition occurred and electron transport rate (ETR) decreased when the light intensity over 740μmol·m-2s-1. The one-year-old leaves had the highest ETR and the most efficiency of light energy, but they also exhibited photoinhibition when the light intensity over 1250μmol·m-2s-1. Two-and three-year-old leaves had similar ETR, and four-year-old leaves were most susceptible to photoinhibition with the lowest ETR.The flower bud differentiation process lasted for about three months from the start of inflorescence primordial differentiation in early July to column and pollinia formation at the end of September under the greenhouse climate condition in North China. The process could be divided into 6 phases: undifferentiation phase, inflorescence primordium differentiation phase, flower differentiation phase, sepal differentiation phase, petal differentiation phase, and column and pollinia differentiation phase. The phases of flower differentiation, column and pollinia differentiation were relatively longer. The new plant finished its growth when sepal differentiation phase began.The flower-bud differentiation was significantly accelerated under the treatment of 25/20℃, unaffected under treatment of 30/25℃, but inhibited under treatment of 35/30℃; The flower sheaths increased faster under 25/20℃than those under 30/25℃after 24 days of treatment. But no difference was observed in leaf area between them. The leaf area and flower sheaths increased slowest under the treatment of 35/30℃; When under a higher temperature in the primary stage of the experiment, the decrease of soluble sugar content in new leaves and new pseudobulbs was more prominent in comparison with old leaves and old pseudobulbs, while the starch content in all organs had a smaller decline. After 18 days of treatment, under the treatment of 35/30℃, the soluble sugar content was higher in the new leaves but lower in new pseudobulbs in comparison with other two treatments. The changes of soluble sugar and starch tended to consistently in all organs under the treatment of 30/25℃. The soluble sugar content in new leaves changed opposite to other organs under the treatment of 25/20℃.The contents of GA3, ZR, ABA were promoted while IAA was inhibited under treatment of 25/20℃and 30/25℃. The reverse trend was observed under the treatment of 35/30℃. The low content of GA3, ZR, ABA and high content of IAA were not good for flower-bud differentiation; The rate of GA3/IAA, GA3/ZR sustained at a stable level under the treatment of 35/30℃which was not good for flower-bud differentiation. It was required lower rate of IAA/ZR and IAA/ABA to keep the flower-bud differentiation continuing.When the different temperature experiment began at undifferentiation phase, it was significantly accelerated the blossom time of Cattleya under the treatment of 25/20℃, which 56 days earlier than that of control, but with lower flowering rate and single flower was dominate. It was not only accelerated the blossom time under the treatment of 25/20℃, but also significantly increased the size of flowers when the experiment began at sepal differentiation phase. The blossom day was 14 days earlier and double flower was dominated. The florescence was inhibited under the treatment of 35/30℃when began at undifferentiation phase, but postponed 14 days when began at sepal differentiation phase. There was no difference between the treatment of 30/25℃and the control. The blossom day would be postponed 36 days to the New Year's Day after the anaphase low temperature treatment of 10/6 ℃on the period of flower buds out of sheathes.It was remarkably advanced the date of florescence and prolonged the length of pedicel and scape after spraying GA3 with 300 mg·kg-1 and 600 mg·kg-1. The blossom date was postponed 6.67 days by spraying 200 mg·kg-1 NAA, but there was no effect on flower quality by spraying different concentration of NAA. The blossom time was 13.34 days and 22.34 days earlier than that of the control by ejecting GA3 with 60 mg·kg-1 and 120 mg·kg-1, at the same time the length of sepal and petal as the same as pedicel and scape were significantly prolonged. The flower size was increased by ejecting NAA with 10 mg·kg-1. There was no influence on florescence date by using ABA, no matter spraying or ejecting, but the flowering rate and size of flowers were decreased when ejecting ABA with 40 mg·kg-1. So ejecting was the efficient way which was recommended to use to regulate the date of floresence. When ejecting 60 mg·kg-1 GA3 or 10 mg·kg-1 NAA, it was not only to advance the floresence date, but also to enlarge the flowers size.Soluble protein content of petals decreased gradually. Membrane permeability, MDA content and O2·- production rates gradually enhanced while SOD and POD activity reduced with the fading of petals. The contents of endogenous ZRs decreased, but IAA,ABA content increased during senescence, ethylene production rate was typical climacteric type, and GA3 content didn't change obviously.Total RNA was extracted from flowers of cattleya, According to the conserved acid sequence for ACC oxidase in other orchids, we designed a pair of oligo nucleotide primers. Using RT-PCR method, a cDNA fragment about 967 base pair which encoded 321 predicted amino acid residues were amplified. The result of BLAST showed the sequence presented a very high match with the ACO genes from the other orchids, the homologue was higher than 85%, especially the original species and relatives which the homologue was higher than 95%. An antisense expression vector of the cattleya-ACO gene which named pBI121ACC was constructed, in which the antisense sequence was controlled by the CaMV 35S promoter.This research provides novel knowledge of growth-development, fertilizer and light requirement of Cattleya under the greenhouse climate condition in North China. It also gives new insights into the flower bud differentiation process, offering theory basics of developing proper cultivation strategies for flowering regulation. It was the first time to reveal the roles of different temperatures in nutrient substances and hormones, and consequently in flower bud differentiation. It was successfully, for the first time, to regulate flowering date for fluffing the demands during the flower fast-selling season by using different temperatures and hormones treatment. An antisense expression vector of the cattleya-ACO gene was constructed which would provide the foundations of the future transgenic research for prolonging the flowering period of the transgenic Cattleya via antisense technology. |
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