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Abstract Using sucrose， citric acid and vitamin C as the basic liquid， different concentrations of 6BA and B9 preservatives were added， and the carnation variety ‘Master’ was treated. The results showed that the formula of 3% sucrose + 50 mg/L citric acid + 600 mg/L vitamin C + 20 mg/L 6BA could effectively maintain the water balance of the cut flower， prolong the vase life of the cut flower to 16.4 d， and maximize the flower diameter and the crown height.
Key words 6BA； B9； Carnation cut flower； Freshkeeping effect
Received： August 20， 2017 Accepted： November 10， 2017
Yanrong BAI （1972-）， male， P. R. China， associate professor， devoted to research about the production of garden flower.
*Corresponding author. Email：[email protected].
Carnation （Dianthus caryophyllus L.）， has beautiful flower shape and color. It has higher ornamental value， and is one of the most important four cut flowers on the international markets， as well as an important material for flower arrangement in daily life[1]. There are many studies on the fresh keeping of carnation cut flower. The preservatives of carnation are generally composed of sucrose， citric acid and vitamin C， and spermidine and benzoic acid， and 1MCP are also used as preservatives. Carnation cut flower are highly sensitive to exogenous ethylene， and the vase life is also affected by microorganisms[2]. Sodium dichloroisocyanurate and white liquor as bactericides could prolong the vase life of carnation cut flower， while the relation between the vessel blocking of the base of the stems and its aging is still not clear. As to plant growth regulators， IAA， GA， 6BA and paclobutrazol are used to keep carnation cut flower fresh[3]， while there were no studies using 6BA and B9 as preservatives for carnation cut flower. Sucrose is one of the main nutrient sources of carnation cut flowers， as well as one of the important components of preservative. Sucrose provides the substrate for the respiration of carnation cut flower， facilitates protein synthesis[4]， and affects water balance， thereby achieving the effects of reducing transpiration and promoting flower opening. Therefore， in preservative， sugar and bactericide， citric acid are applied in combination[5]， which could enhance the water absorping capacity of vascular bundles of flower stems， increase the fresh weight of cut flower， and retard the reaching of the balance value， thereby prolonging the vase life[6]. B9 is a kind of growth retardant， which could postpone the opening of flower and prolong the vase life of cut flower. 6BA is 6benzylaminopurine， with a molecular formula： C12H11N5. As a plant growth regulator， 6BA has no side effects， which can promote cell division， delay chlorophyll degradation， reduce the sensitivity of cut flower to ethylene， and inhibit the generation of ethylene， thereby delaying the aging of flower[7]. In this study， B9 and 6BA were combined to investigate the freshkeeping and antiaging effects of B9 and 6BA combination on carnation cut flower through the comprehensive assessment of carnation shape， longevity and water balance value， change rate of fresh weight， vase life of carnation cut flower， flower diameter， and crown height， so as to select an economic， safe and effective preservative formula[8]. The study will provide a theoretical basis for the improvement of the freshkeeping effect on carnation cut flower. Materials and Methods
Experimental materials
The tested material was ‘Master’， provided by the Flowergrowing Center of Yunnan Academy of Agricultural Sciences. Strong flower shoots free of diseases and pests with basically the same opening of flowers and a stem length of 80-90 cm were cut diagonally （45°） before culture in vase， and the cut flowers had a length ranging from 35 to 40 cm， on each of which three leaves were left.
Methods
The experiments were designed with five treatments in total： T1， T2， T3， T4， and CK （distilled water）. Each treatment had three replicates， and each vase included three shoots. The flower shoots were vertically inserted in above solutions， and the bases were immersed with a length of 5 cm. The flower shoots were cultured in vases in laboratory at a temperature of 15-20 ℃ under a relative humidity of 80%. After culture at （15±2） ℃ for one day， the flower shoots were taken out， and the bases were cut off at the position of 1 cm. The flower shoots were then cultured in conical flasks filled with 250 ml of clear water， and each vase included three shoots. The openings of the conical flasks were sealed with cotton to prevent water evaporation， and redundant lateral buds were cut off， leaving only one bud to ensure normal vegetative growth and reproductive growth， so as avoid experiment error. During the preparation of preservative， attention should be paid to the measurement of various elements. Citric acid should be prepared freshly for use， and during the preparation， the solution should be stirred with glass rod to ensure sufficient dissolution.
Table 1 Preservative formula for carnation ‘Master’
TreatmentPreservative formula
T13% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +20 mg/L 6BA
T23% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +200 mg/L B9
T33% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +20 mg/L 6BA+200 mg/L B9
T43% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +50 mg/L 6BA+300 mg/L B9
CKDistilled water
Determination of observation indexes and data processing
Determination of vase life of cut flower
The determination was started from the culture in clear water. Every day， the appearance and the opening of the flowers were observed. The wilting degree of the petals was observed. When the flower wilted seriously， the petals were dried and wrinkled， and the color of the flowers became dark due to serious water loss， accompanied by dehydration of the stem and bending of the flower. At this time， the carnation lost its socalled ornamental value， which also means that the vase life of the cut flower has already over. Determination of physiological indexes of carnation cut flower
The various indexes were determined from inserting the carnation cut flower into various treatment solutions. At first， the sum of the weights of the flower shoot， the liquid and the conical flask was measured as F0. Then， the carnation cut flower was taken out， and the sum of the weights of the liquid and the conical flask was measured as T0. The sum of the weights of the flower shoot， the liquid and the conical flask and the sum of the weights of the liquid and the conical flask were measured every other day as Ft （t=1， 2， 3…） and Tt， respectively. The calculation was performed according to following equations： Water loss of flower shoot = （Ft-1- Tt）； Water absorbing capacity of flower shoot = （Ft-1-Ft）； Water balance value = （Water absorbing capacity-Water loss）； Fresh weight = （Ft-Tt）； and Relative fresh weight = （Ft -Tt）/（F0-T0）.
Determination of flower diameter and crown height of carnation ‘Master’
The flower diameter and crown height were determined from the culture in clear water. The flower diameter and crown height were recorded after the measurement with a vernier caliper， until the end of the vase life.
Results and Analysis
Effects of different freshkeeping solutions on the flower diameter of carnation ‘Master’
The flower diameter of the cut flower is an assessment index of the ornamental value of the cut flower. As shown in Table 1， on the 0th d of treatment， different formulas had no significant effects on the flower diameter of the carnation cut flower， and there were no significant differences between various treatments. On the 2nd d， there were still no significant differences between various treatments， and from the 0th d to the 2nd d， the flower diameter of the carnation cut flower was increasing continuously. On the 4th d， still no significant differences were observed between various treatments， but apparently from the flower diameter of various treatment， treatment T1 exhibited the largest flower diameter， followed by treatment T2， and the order was T1>T2>T3>T4>CK. On the 6th d， there were no significant differences between various treatments， and it could be seen that treatment T1 had the largest flower diameter， followed by treatment T2， and the flower diameters were in order of T1>T2>T3>T4>CK. On the 8th d， there was a significant difference between T1 and distilled water （CK）. Specifically， the flower diameter of T1 was higher than the CK by 63.2%， but was not significantly different from those of treatments T2， T3 and T4； there were no significant differences between treatments T2， T3 and T4 and the CK； and the flower diameter changed continuously， and the flower diameter of treatment T1 reached the maximum value. On the 10th d， there were no significant differences between various treatments， and over time， the flower diameter decreased. With the prolonging of the culture days， the flower diameter of treatment T1 increased continuously， and reached the maximum value of 9.62 cm on the 8th d， and the flower began to wilt on the 10th d. Treatment T3 exhibited the maximum value of 6.09 cm on the 6th d， and the flower wilted on the 8th d， followed by the continuous decrease of flower diameter. Treatment T4 showed the maximum value of 5.77 cm on the 6th d， and the flower began to wilt on the 8th d， and nearly withered and fell on the 10th d. It could be seen from the table that the vase value of flower in the CK was over on the 10th d， and the flower diameter changed little. Effects of different treatments on crown height of carnation cut flower
The crown height of the cut flower is an assessment index of the ornamental value of the cut flower. As shown in Table 2， on the 0th d， treatment T3 had no significant difference from the CK in crown height， while there was a significant difference from treatment T2 and the CK， i.e.， the crown height of treatment was significantly higher than that of the CK， with a difference of 28.57%. There was also a significant difference between treatment T4 and CK， i.e.， the crown diameter of T4 was significantly lower than that of the CK， with a difference of 18.01%. On the 2nd d， different concentrations of 6BA and B9 had a greater effect on the crown height of carnation cut flower. Specifically， the crown height of treatment T1 was significantly different from that of the CK， i.e.， higher than that of the CK by 20.75%； treatment T2 had a crown height significantly higher than that of the CK， higher than that of the CK by 30.22%； treatment T3 was not significantly different from the CK； and the crown height of treatment T4 was significantly lower than that of the CK， with a difference of 20.15%. On the 3rd d， treatments T1， T2， T3 and T4 had no significant differences from the CK in crown height. On the 6th d， different concentrations of 6BA and B9 showed a greater effect on the crown height of carnation. Specifically， the crown height of treatment T1 was significantly higher than that of CK， by 17.4%； the value of treatment T2 was significantly higher than that of the CK， with a difference of 18.5%； treatment T3 exhibited a crown height significantly higher than that of the CK by 15.96%； and the crown height of treatment T4 was significantly lower than that of the CK. On the 8th d， different concentrations of 6BA and B9 exhibited a greater effect on the crown height of carnation. Specifically， the crown height of treatment T1 was lower than that of the CK， with a difference of 24.82%； treatment T2 showed a crown height significantly lower than that of the CK， with a difference of 27.24%； and treatment T3 had a crown height significantly lower than that of the CK， with a difference of 20.24%. On the 10th d， different concentrations of 6BA and B9 showed a greater effect on the crown height of carnation. Specifically， treatment T1 showed a crown height significantly higher than that of the CK， with a difference of 31.21%； the crown height of treatment T2 was significantly higher than that of the CK， with a difference of 26.83%； treatment T3 exhibited a value significant higher than that of the CK， with a difference of 20.54%； and treatment T4 had no significant difference from the CK in crown height. Yanrong BAI et al. Freshkeeping Effects of Different Concentrations of 6BA and B9 on Carnation Cut Flower
Table 2 Comparison of the diameter of carnation flower under different treatments
Treatment0 d2 d4 d6 d8 d 10 d
T16.27±5.75 a7.45±1.05 a9.38±6.10 a9.59±6.25 a9.62±5.81 a8.89±4.86 a
T24.92±0.19 a5.88±0.33 ab5.94±0.34 a6.24±0.20 a6.46±0.12 ab6.21±0.67 a
T33.60±1.44 a4.58±1.6 ab5.64±0.70 a6.09±0.48 a6.07±0.68 ab5.62±1.27 a
T41.83±0.22 a2.85±0.27 b4.73±0.43 a5.77±0.15 a5.60±0.76 ab1.27±0.17 a
CK3.45±1.05 a4.52±1.03 ab4.51±0.07 a4.71±0.01 a3.54±0.19 b-
- represents that the vase life of the cut flower is over.
Table 3 Comparison of crown height of carnation cut flower under different treatments
Treatment0 d2 d4 d6 d8 d10 d
T11.56±0.06 b1.92±0.04 b2.08±0.19 a2.24±0.18 a2.46±0.23 a2.70±0.19 a
T22.07±0.07 a1.82±0.04 b2.08±0.19 a2.27±0.09 a2.54±0.06 a2.54±0.15 ab
T31.58±0.03 b1.68±0.29 bc2.08±0.19 a2.20±0.19 a2.32±0.13 ab2.34±0.26 bc
T41.32±0.14 c1.27±0.05 d1.54±0.14 b1.84±0.01 b2.04±0.31 bc2.07±0.01 cd
CK1.61±0.01 b1.59±0.16 c1.93±0.26 ab1.85±0.12 b1.85±0.01 c1.86±0.03 d
Effects on flower weight under different treatments
It could be seen from Table 3 that on the 0th d， different concentrations of 6BA and B9 had a greater effect on the flower weight of carnation cut flower. Specifically， the flower weight of treatment T1 was significantly lower than that of the CK， with a difference of 14.35%； treatment T2 showed a value significantly lower than that of the CK， with a difference of 7.12%； flower weight of treatment T3 had no significant difference from the CK； and the value of treatment T4 was lower than that of the CK. On the 2nd d， different concentrations of 6BA and B9 had a greater effect on the flower weight of carnation cut flower. Specifically， the flower weight of treatment T1 was significantly lower than that of the CK， with a difference of 7.55%； there was no significant difference between treatment T2 and the CK； the flower weight of treatment T3 was also not significantly different from that of the CK； and treatment T4 exhibited a flower weight significantly lower than that of the CK， by 15.67%. On the 4th d， different concentrations of 6BA and B9 had no high effect on the flower weight of carnation cut flower. Specifically， under treatment T1 and the CK， there was no significant difference in the flower weight of carnation cut flower； treatment T2 was not significantly different from the CK in flower weight； similarly， treatment T3 was not significantly different from the CK in flower weight； and the flower weight of treatment T4 was significantly lower than that of the CK， with a difference of 10.33%. On the 6th d， specifically， there was no significant difference between treatment T1 and the CK； the flower weight of treatment T1 was higher than that of the CK， with a difference of 9.44%； the value of treatment T3 was significantly higher than that of the CK， with a difference of 2.41%； and there was no significant difference between treatment T4 and the CK. On the 8th d， specifically， treatment T1 had no significant difference from the CK in flower weight； treatment T2 also had no significant difference from the CK； the flower weight of treatment T3 was significantly higher than that of the CK， with a difference of 14.76%； and there was no significant difference between treatment T4 and the CK. On the 10th d， specifically， treatments T1， T2， T3 and T4 were all not significantly different from the CK in flower weight. Effects of different solution formula on the ornamental value of carnation cut flower
The maximum flower diameter and maximum crown height are both the assessment indexes of the ornamental value of carnation cut flower. It could be seen from Table 4 that the carnation cut flower in the CK only had a vase life of 7 d. Different solution treatments greatly affected the vase life of the carnation cut flower. Specifically， treatment T1 was significantly different from the CK， with a difference of 57.3%， and it was the treatment with the longest vase life among the various treatments； the vase life of treatment T2 was significantly higher than the CK， with a difference of 54.2%； treatment T3 was not significantly different from the CK in vase life； and the vase life of treatment T4 was significantly higher than the CK， with a difference of 50.3%. So the vase life showed an order of T1>T2>T3>T4>CK， and the mother liquid and 20 mg/L 6BA facilitated the prolonging of the vase life of carnation cut flower. Treatment T1 was significant different from the CK in the maximum flower diameter， i.e.， significantly higher than the CK by 56.26%； treatment T2 was not significantly different from the CK； treatment T3 was significantly higher than the CK in the maximum flower diameter， significantly higher by 39.35%； and treatment T4 was significantly higher than the CK， significantly higher by 43.85%. Comprehensively from various treatments， the flower diameters were in order of T1>T2>T3>T4>CK. As to the maximum crown height， treatment A was significantly higher than the CK， significantly higher by 55.8%； treatment T2 was significantly higher than the CK， significantly higher by 46.33%； treatment T3 was significant higher than the CK， by 33.65%； and treatment T4 was significantly higher than the CK， with a difference of 44.2%. The maximum crown heights were in order of T1>T2>T3>T4>CK. It could be seen from Table 4 that treatment T1 was better among all the treatments， whether for vase life or flower diameter and crown height. Comprehensively， the ornamental value of the various treatments was in order of T1>T2>T3>T4>CK.
Table 4 Comparison of flower weight of carnation cut flower under different treatment
Treatment0 d2 d4 d 6 d 8 d 10 d
T112.42±0.18 c13.91±0.19 b14.37±0.17 bc15.19±0.70 ab15.18±0.89 ab13.78±0.55 ab
T213.47±0.46 b14.94±0.57 a15.52±0.59 a15.86±0.70 a15.42±1.33 ab14.46±0.82 b
T312.76±0.25 ab14.79±0.23 a15.61±0.03 a15.94±0.23 a15.75±1.24 a15.19±1.76 a T412.22±0.54 c13.49±0.61 b13.98±0.74 c14.40±0.70 b14.61±0.68 ab13.44±0.37 b
CK14.49±0.26 a15.04±0.19 a15.08±0.07 ab14.37±0.19 b13.44±0.37 b12.87±0.1 ab
Table 5 Comparison of ornamental value of carnation cut flower under different solution treatments
TreatmentVase life∥dMaximum flower diameter∥cmMaximum crown height∥cm
T116.4±11.8 ab7.12±6.77 c4.48±4.23 ab
T215.3±12.6 bc6.88±5.12 ad3.69±2.82 c
T311.8±9.7 d4.32 ±3.88 b2.98±2.22 bc
T414.1±9.8 c5.55 ±4.85 c3.55±3.11 a
CK7±5.6 d3.12±2.66 d1.98±1.68 d
Effects of different formula solutions on the water balance value of carnation cut flower
The effects of different formula solutions on the water balance value of carnation cut flower are shown in Fig. 1. On the 0th d， no treatments remarkably promoted the water absorption of carnation， and the positive water balance value indicated that the cut flower absorbed more water than the water it lost. On the 2nd d， treatment T3 and the CK， T1， T4 and T2 all absorbed water， and compared with the 1st d， the carnation cut flower absorbed water continuously. On the 4th d， the water balance value of treatment T1 reached the maximum value， i.e.， the water absorbing capacity reached the maximum value， while the water absorbing capacity of the CK decreased continuously， and the cut flower in the CK began to wilt. A negative water balance value indicates the cut flower loses more water than the water it absorbs， so the flower will have a tendency of wilting. On the 4th d， the water absorbing capacity of treatment T3 reached its maximum value， the water absorbing capacity values of treatments T4 and T2 were also the largest than the values on the 0th d and the 2nd d， while the CK exhibited a negative value， indicating that the cut flower began to lose water. On the 6th d， the water absorbing capacity of the CK decreased， and the cut flower wilted， indicating that the CK suffered serious water loss. On the 8th d， treatment T1 began to absorb water slowly， and was measured with an error， while treatment T2 and treatments T3， T1 and T4 wilted slowly， and the CK was suffering increasingly serious water loss. Since the 10th d， the wilting of various treatments became more and more serious. The formula solution of treatment T1 was the one capable of prolonging the vase life of cut flower
Fig. 1 Effects of different solutions on water balance value of carnation cut flower
Fig. 2 Variation of relative fresh weight of carnation cut flower under different treatments among all the treatments. It could be seen from the figure that 6BA and B9 could remarkably improve the water condition in flower body， and retarded the wilting process caused by water loss.
Variation of relative fresh weight of cut flower under different treatments
For the effects of different preservative solutions on the relative fresh weight of carnation cut flower （Fig. 2）， within certain culture time， the relative fresh weights of treatments T1， T2， T3 and T4 were higher than that of the CK， and from the 0th d to the 4th d， under the different preservative treatments， the relative fresh weight of the carnation cut flower increased continuously， i.e.， the water absorption of carnation was promoted. The relative fresh weight of treatment T1 increased from the beginning of the experiment， and decreased from the 8th d. The relative fresh weight of treatment T2 began to decrease on the 8th d， and the carnation wilted， indicating that its vase life was going to be ended. The relative fresh weight of treatment T3 began to decrease on the 8th d， indicating that the carnation cut flower began to wilt， the vase life was going to be ended. However， the relative fresh weight of the CK decreased continuously， and the vase life was also on the decrease. Treatment T4 exhibited a decreasing trend from the 4th d.
Discussion
In this study， B9 is a kind of antiaging growth regulator， which has bactericidal effect and is applied widely. It could serve as dwarfing agent， fruit setting agent， rooting agent and preservative， and could simultaneously delay the opening of flower buds and retard aging. It could slow down the growth of flower after collection to a certain degree， prevent the biosynthesis of gibberellic acid in tissue and its metabolic process through the inhibition of plant growth， and improve the tolerance of cut flower against stress， thereby prolonging the vase life of cut flower. 6BA could prolong the vase life of carnation cut flower by affecting the water absorption of carnation cut flower and reducing respiratory rate. Zheng et al.[4] studied the physiological effect of 6BA and kinetin against the aging of carnation cut flower. In the study， 3% surcose+200 mg/L 8hydroxyquinoline + 200 mg/L citric acid +50 mg/L 6BA could prolong the vase life of cut flower to 23.5 d， while 3% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +20 mg/L 6BA in this study could prolong the vase life of carnation to 16.4 d， which is shorter than that in previous studies. Therefore， 8hydroxyquinoline could remarkably prolong the vase life of cut flower， and the solution formula in the experiment still should be improved. Zeng et al.[8] studied the freshkeeping effect of the combined application of CaCl2 and B9 on carnation cut flower， and the combination of 250 mg/L B9 and 0.1 % CaCl2 exhibited the best freshkeeping effect， under which the vase life and fresh degree of carnation were the best. In the study， treatment T2 used 200 mg/L B9， with which the flower diameter and crown height of the cut flower increased remarkably， so the B9 concentration in the solution was proper. Yuan et al.[7] investigated the freshkeeping effect of environmentfriendly freshkeeping solution on carnation cut flower. The results showed that 7.5% sucrose +2.5 mg/L citric acid +2.5 mg/L 8HQ had the best treatment effect. The solution had remarkably antiaging and freshkeeping effects on carnation cut flower. Specifically， it could remarkably prolong the vase life of carnation cut flower and increase the water curing time by 20 d. This formula prolonged the vase life of carnation cut flower， but the fresh weight had no remarkable change. The comparison of this study with previous studies indicates further study is still needed， so as to find a formula which not only could prolong the vase life of cut flower， but also could improve the fresh degree of cut flower. Conclusion
In this study， the four pretreatment solutions could prolong the vase life of carnation cut flower to different degrees， and also could achieve of the effects of increasing flower fresh weight and flower diameter as well as crown height， promoting flowering and keeping water balance. Among all the treatments， treatment T1， 3% sucrose + 50 mg/L citric acid +600 mg/L vitamin C+20 mg/L 6BA had the best effect， and the vase life of carnation cut flower reached its maximum value of 16.4 d. Treatment T1 is a preferable formula with good stability and good safety， which could remarkably improve economic and ornamental value of carnation cut flower.
References
[1] LUO HY， JING HJ， LI JR， et al. Effects of different preservatives on fresh keeping of cut carnation flower[J]. Plant Physiology Communications， 2003， 39（1）： 27-28.
[2] ZHEN ZX， ZHANG JL， LI YH， et al. Effect of some freshness keeping chemicals on cut carnation[J]. Hebei Journal of Forestry and Orchard Research， 2007， 22（3）： 321-323.
[3] LUO HY， JING HJ， WANG FY， et al. The influence of inorganic salts on fresh preservation of cut carnation flower[J]. Journal of Huazhong Normal University： Natural Sciences， 2003， 37（1）： 99-101.
[4] ZHENG CP， WU D， LI L， et al. Physiological effects of 6BA and KT on senescence of cut carnation （Dianthus caryophyllus L.） flowers[J]. Plant Physiology Communications， 2008，44（6）：1152-1154.
[5] ZHAO M， ZHU DQ， WANG JY， et al. Preservation of carnation cut flowers dealt with preservative solution of different formulations[J]. Guizhou Agricultural Sciences， 2014， （6）： 144-146.
[6] ZHAO M， CHEN CG， WANG LM， et al. Effect of different preservatives on physiological and biochemical characteristics of cut carnation flower[J]. Hubei Agricultural Sciences， 2009， 48（7）： 1680-1682.
[7] YUAN JH. Preservative Effect of Environmentfriendly preservative solution on cut carnation flower [J]. Hubei Agricultural Sciences， 2013， 52（16）： 3926-3929.
[8] ZENG CL， CHANG SW， BIAN BW， et al. Effect of CaCl2 combining with B9 on the fresh preservation of cut carnation flowers[J]. Journal of Anhui Agricultural Sciences， 2007， 35（2）： 333-335.
Key words 6BA； B9； Carnation cut flower； Freshkeeping effect
Received： August 20， 2017 Accepted： November 10， 2017
Yanrong BAI （1972-）， male， P. R. China， associate professor， devoted to research about the production of garden flower.
*Corresponding author. Email：[email protected].
Carnation （Dianthus caryophyllus L.）， has beautiful flower shape and color. It has higher ornamental value， and is one of the most important four cut flowers on the international markets， as well as an important material for flower arrangement in daily life[1]. There are many studies on the fresh keeping of carnation cut flower. The preservatives of carnation are generally composed of sucrose， citric acid and vitamin C， and spermidine and benzoic acid， and 1MCP are also used as preservatives. Carnation cut flower are highly sensitive to exogenous ethylene， and the vase life is also affected by microorganisms[2]. Sodium dichloroisocyanurate and white liquor as bactericides could prolong the vase life of carnation cut flower， while the relation between the vessel blocking of the base of the stems and its aging is still not clear. As to plant growth regulators， IAA， GA， 6BA and paclobutrazol are used to keep carnation cut flower fresh[3]， while there were no studies using 6BA and B9 as preservatives for carnation cut flower. Sucrose is one of the main nutrient sources of carnation cut flowers， as well as one of the important components of preservative. Sucrose provides the substrate for the respiration of carnation cut flower， facilitates protein synthesis[4]， and affects water balance， thereby achieving the effects of reducing transpiration and promoting flower opening. Therefore， in preservative， sugar and bactericide， citric acid are applied in combination[5]， which could enhance the water absorping capacity of vascular bundles of flower stems， increase the fresh weight of cut flower， and retard the reaching of the balance value， thereby prolonging the vase life[6]. B9 is a kind of growth retardant， which could postpone the opening of flower and prolong the vase life of cut flower. 6BA is 6benzylaminopurine， with a molecular formula： C12H11N5. As a plant growth regulator， 6BA has no side effects， which can promote cell division， delay chlorophyll degradation， reduce the sensitivity of cut flower to ethylene， and inhibit the generation of ethylene， thereby delaying the aging of flower[7]. In this study， B9 and 6BA were combined to investigate the freshkeeping and antiaging effects of B9 and 6BA combination on carnation cut flower through the comprehensive assessment of carnation shape， longevity and water balance value， change rate of fresh weight， vase life of carnation cut flower， flower diameter， and crown height， so as to select an economic， safe and effective preservative formula[8]. The study will provide a theoretical basis for the improvement of the freshkeeping effect on carnation cut flower. Materials and Methods
Experimental materials
The tested material was ‘Master’， provided by the Flowergrowing Center of Yunnan Academy of Agricultural Sciences. Strong flower shoots free of diseases and pests with basically the same opening of flowers and a stem length of 80-90 cm were cut diagonally （45°） before culture in vase， and the cut flowers had a length ranging from 35 to 40 cm， on each of which three leaves were left.
Methods
The experiments were designed with five treatments in total： T1， T2， T3， T4， and CK （distilled water）. Each treatment had three replicates， and each vase included three shoots. The flower shoots were vertically inserted in above solutions， and the bases were immersed with a length of 5 cm. The flower shoots were cultured in vases in laboratory at a temperature of 15-20 ℃ under a relative humidity of 80%. After culture at （15±2） ℃ for one day， the flower shoots were taken out， and the bases were cut off at the position of 1 cm. The flower shoots were then cultured in conical flasks filled with 250 ml of clear water， and each vase included three shoots. The openings of the conical flasks were sealed with cotton to prevent water evaporation， and redundant lateral buds were cut off， leaving only one bud to ensure normal vegetative growth and reproductive growth， so as avoid experiment error. During the preparation of preservative， attention should be paid to the measurement of various elements. Citric acid should be prepared freshly for use， and during the preparation， the solution should be stirred with glass rod to ensure sufficient dissolution.
Table 1 Preservative formula for carnation ‘Master’
TreatmentPreservative formula
T13% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +20 mg/L 6BA
T23% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +200 mg/L B9
T33% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +20 mg/L 6BA+200 mg/L B9
T43% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +50 mg/L 6BA+300 mg/L B9
CKDistilled water
Determination of observation indexes and data processing
Determination of vase life of cut flower
The determination was started from the culture in clear water. Every day， the appearance and the opening of the flowers were observed. The wilting degree of the petals was observed. When the flower wilted seriously， the petals were dried and wrinkled， and the color of the flowers became dark due to serious water loss， accompanied by dehydration of the stem and bending of the flower. At this time， the carnation lost its socalled ornamental value， which also means that the vase life of the cut flower has already over. Determination of physiological indexes of carnation cut flower
The various indexes were determined from inserting the carnation cut flower into various treatment solutions. At first， the sum of the weights of the flower shoot， the liquid and the conical flask was measured as F0. Then， the carnation cut flower was taken out， and the sum of the weights of the liquid and the conical flask was measured as T0. The sum of the weights of the flower shoot， the liquid and the conical flask and the sum of the weights of the liquid and the conical flask were measured every other day as Ft （t=1， 2， 3…） and Tt， respectively. The calculation was performed according to following equations： Water loss of flower shoot = （Ft-1- Tt）； Water absorbing capacity of flower shoot = （Ft-1-Ft）； Water balance value = （Water absorbing capacity-Water loss）； Fresh weight = （Ft-Tt）； and Relative fresh weight = （Ft -Tt）/（F0-T0）.
Determination of flower diameter and crown height of carnation ‘Master’
The flower diameter and crown height were determined from the culture in clear water. The flower diameter and crown height were recorded after the measurement with a vernier caliper， until the end of the vase life.
Results and Analysis
Effects of different freshkeeping solutions on the flower diameter of carnation ‘Master’
The flower diameter of the cut flower is an assessment index of the ornamental value of the cut flower. As shown in Table 1， on the 0th d of treatment， different formulas had no significant effects on the flower diameter of the carnation cut flower， and there were no significant differences between various treatments. On the 2nd d， there were still no significant differences between various treatments， and from the 0th d to the 2nd d， the flower diameter of the carnation cut flower was increasing continuously. On the 4th d， still no significant differences were observed between various treatments， but apparently from the flower diameter of various treatment， treatment T1 exhibited the largest flower diameter， followed by treatment T2， and the order was T1>T2>T3>T4>CK. On the 6th d， there were no significant differences between various treatments， and it could be seen that treatment T1 had the largest flower diameter， followed by treatment T2， and the flower diameters were in order of T1>T2>T3>T4>CK. On the 8th d， there was a significant difference between T1 and distilled water （CK）. Specifically， the flower diameter of T1 was higher than the CK by 63.2%， but was not significantly different from those of treatments T2， T3 and T4； there were no significant differences between treatments T2， T3 and T4 and the CK； and the flower diameter changed continuously， and the flower diameter of treatment T1 reached the maximum value. On the 10th d， there were no significant differences between various treatments， and over time， the flower diameter decreased. With the prolonging of the culture days， the flower diameter of treatment T1 increased continuously， and reached the maximum value of 9.62 cm on the 8th d， and the flower began to wilt on the 10th d. Treatment T3 exhibited the maximum value of 6.09 cm on the 6th d， and the flower wilted on the 8th d， followed by the continuous decrease of flower diameter. Treatment T4 showed the maximum value of 5.77 cm on the 6th d， and the flower began to wilt on the 8th d， and nearly withered and fell on the 10th d. It could be seen from the table that the vase value of flower in the CK was over on the 10th d， and the flower diameter changed little. Effects of different treatments on crown height of carnation cut flower
The crown height of the cut flower is an assessment index of the ornamental value of the cut flower. As shown in Table 2， on the 0th d， treatment T3 had no significant difference from the CK in crown height， while there was a significant difference from treatment T2 and the CK， i.e.， the crown height of treatment was significantly higher than that of the CK， with a difference of 28.57%. There was also a significant difference between treatment T4 and CK， i.e.， the crown diameter of T4 was significantly lower than that of the CK， with a difference of 18.01%. On the 2nd d， different concentrations of 6BA and B9 had a greater effect on the crown height of carnation cut flower. Specifically， the crown height of treatment T1 was significantly different from that of the CK， i.e.， higher than that of the CK by 20.75%； treatment T2 had a crown height significantly higher than that of the CK， higher than that of the CK by 30.22%； treatment T3 was not significantly different from the CK； and the crown height of treatment T4 was significantly lower than that of the CK， with a difference of 20.15%. On the 3rd d， treatments T1， T2， T3 and T4 had no significant differences from the CK in crown height. On the 6th d， different concentrations of 6BA and B9 showed a greater effect on the crown height of carnation. Specifically， the crown height of treatment T1 was significantly higher than that of CK， by 17.4%； the value of treatment T2 was significantly higher than that of the CK， with a difference of 18.5%； treatment T3 exhibited a crown height significantly higher than that of the CK by 15.96%； and the crown height of treatment T4 was significantly lower than that of the CK. On the 8th d， different concentrations of 6BA and B9 exhibited a greater effect on the crown height of carnation. Specifically， the crown height of treatment T1 was lower than that of the CK， with a difference of 24.82%； treatment T2 showed a crown height significantly lower than that of the CK， with a difference of 27.24%； and treatment T3 had a crown height significantly lower than that of the CK， with a difference of 20.24%. On the 10th d， different concentrations of 6BA and B9 showed a greater effect on the crown height of carnation. Specifically， treatment T1 showed a crown height significantly higher than that of the CK， with a difference of 31.21%； the crown height of treatment T2 was significantly higher than that of the CK， with a difference of 26.83%； treatment T3 exhibited a value significant higher than that of the CK， with a difference of 20.54%； and treatment T4 had no significant difference from the CK in crown height. Yanrong BAI et al. Freshkeeping Effects of Different Concentrations of 6BA and B9 on Carnation Cut Flower
Table 2 Comparison of the diameter of carnation flower under different treatments
Treatment0 d2 d4 d6 d8 d 10 d
T16.27±5.75 a7.45±1.05 a9.38±6.10 a9.59±6.25 a9.62±5.81 a8.89±4.86 a
T24.92±0.19 a5.88±0.33 ab5.94±0.34 a6.24±0.20 a6.46±0.12 ab6.21±0.67 a
T33.60±1.44 a4.58±1.6 ab5.64±0.70 a6.09±0.48 a6.07±0.68 ab5.62±1.27 a
T41.83±0.22 a2.85±0.27 b4.73±0.43 a5.77±0.15 a5.60±0.76 ab1.27±0.17 a
CK3.45±1.05 a4.52±1.03 ab4.51±0.07 a4.71±0.01 a3.54±0.19 b-
- represents that the vase life of the cut flower is over.
Table 3 Comparison of crown height of carnation cut flower under different treatments
Treatment0 d2 d4 d6 d8 d10 d
T11.56±0.06 b1.92±0.04 b2.08±0.19 a2.24±0.18 a2.46±0.23 a2.70±0.19 a
T22.07±0.07 a1.82±0.04 b2.08±0.19 a2.27±0.09 a2.54±0.06 a2.54±0.15 ab
T31.58±0.03 b1.68±0.29 bc2.08±0.19 a2.20±0.19 a2.32±0.13 ab2.34±0.26 bc
T41.32±0.14 c1.27±0.05 d1.54±0.14 b1.84±0.01 b2.04±0.31 bc2.07±0.01 cd
CK1.61±0.01 b1.59±0.16 c1.93±0.26 ab1.85±0.12 b1.85±0.01 c1.86±0.03 d
Effects on flower weight under different treatments
It could be seen from Table 3 that on the 0th d， different concentrations of 6BA and B9 had a greater effect on the flower weight of carnation cut flower. Specifically， the flower weight of treatment T1 was significantly lower than that of the CK， with a difference of 14.35%； treatment T2 showed a value significantly lower than that of the CK， with a difference of 7.12%； flower weight of treatment T3 had no significant difference from the CK； and the value of treatment T4 was lower than that of the CK. On the 2nd d， different concentrations of 6BA and B9 had a greater effect on the flower weight of carnation cut flower. Specifically， the flower weight of treatment T1 was significantly lower than that of the CK， with a difference of 7.55%； there was no significant difference between treatment T2 and the CK； the flower weight of treatment T3 was also not significantly different from that of the CK； and treatment T4 exhibited a flower weight significantly lower than that of the CK， by 15.67%. On the 4th d， different concentrations of 6BA and B9 had no high effect on the flower weight of carnation cut flower. Specifically， under treatment T1 and the CK， there was no significant difference in the flower weight of carnation cut flower； treatment T2 was not significantly different from the CK in flower weight； similarly， treatment T3 was not significantly different from the CK in flower weight； and the flower weight of treatment T4 was significantly lower than that of the CK， with a difference of 10.33%. On the 6th d， specifically， there was no significant difference between treatment T1 and the CK； the flower weight of treatment T1 was higher than that of the CK， with a difference of 9.44%； the value of treatment T3 was significantly higher than that of the CK， with a difference of 2.41%； and there was no significant difference between treatment T4 and the CK. On the 8th d， specifically， treatment T1 had no significant difference from the CK in flower weight； treatment T2 also had no significant difference from the CK； the flower weight of treatment T3 was significantly higher than that of the CK， with a difference of 14.76%； and there was no significant difference between treatment T4 and the CK. On the 10th d， specifically， treatments T1， T2， T3 and T4 were all not significantly different from the CK in flower weight. Effects of different solution formula on the ornamental value of carnation cut flower
The maximum flower diameter and maximum crown height are both the assessment indexes of the ornamental value of carnation cut flower. It could be seen from Table 4 that the carnation cut flower in the CK only had a vase life of 7 d. Different solution treatments greatly affected the vase life of the carnation cut flower. Specifically， treatment T1 was significantly different from the CK， with a difference of 57.3%， and it was the treatment with the longest vase life among the various treatments； the vase life of treatment T2 was significantly higher than the CK， with a difference of 54.2%； treatment T3 was not significantly different from the CK in vase life； and the vase life of treatment T4 was significantly higher than the CK， with a difference of 50.3%. So the vase life showed an order of T1>T2>T3>T4>CK， and the mother liquid and 20 mg/L 6BA facilitated the prolonging of the vase life of carnation cut flower. Treatment T1 was significant different from the CK in the maximum flower diameter， i.e.， significantly higher than the CK by 56.26%； treatment T2 was not significantly different from the CK； treatment T3 was significantly higher than the CK in the maximum flower diameter， significantly higher by 39.35%； and treatment T4 was significantly higher than the CK， significantly higher by 43.85%. Comprehensively from various treatments， the flower diameters were in order of T1>T2>T3>T4>CK. As to the maximum crown height， treatment A was significantly higher than the CK， significantly higher by 55.8%； treatment T2 was significantly higher than the CK， significantly higher by 46.33%； treatment T3 was significant higher than the CK， by 33.65%； and treatment T4 was significantly higher than the CK， with a difference of 44.2%. The maximum crown heights were in order of T1>T2>T3>T4>CK. It could be seen from Table 4 that treatment T1 was better among all the treatments， whether for vase life or flower diameter and crown height. Comprehensively， the ornamental value of the various treatments was in order of T1>T2>T3>T4>CK.
Table 4 Comparison of flower weight of carnation cut flower under different treatment
Treatment0 d2 d4 d 6 d 8 d 10 d
T112.42±0.18 c13.91±0.19 b14.37±0.17 bc15.19±0.70 ab15.18±0.89 ab13.78±0.55 ab
T213.47±0.46 b14.94±0.57 a15.52±0.59 a15.86±0.70 a15.42±1.33 ab14.46±0.82 b
T312.76±0.25 ab14.79±0.23 a15.61±0.03 a15.94±0.23 a15.75±1.24 a15.19±1.76 a T412.22±0.54 c13.49±0.61 b13.98±0.74 c14.40±0.70 b14.61±0.68 ab13.44±0.37 b
CK14.49±0.26 a15.04±0.19 a15.08±0.07 ab14.37±0.19 b13.44±0.37 b12.87±0.1 ab
Table 5 Comparison of ornamental value of carnation cut flower under different solution treatments
TreatmentVase life∥dMaximum flower diameter∥cmMaximum crown height∥cm
T116.4±11.8 ab7.12±6.77 c4.48±4.23 ab
T215.3±12.6 bc6.88±5.12 ad3.69±2.82 c
T311.8±9.7 d4.32 ±3.88 b2.98±2.22 bc
T414.1±9.8 c5.55 ±4.85 c3.55±3.11 a
CK7±5.6 d3.12±2.66 d1.98±1.68 d
Effects of different formula solutions on the water balance value of carnation cut flower
The effects of different formula solutions on the water balance value of carnation cut flower are shown in Fig. 1. On the 0th d， no treatments remarkably promoted the water absorption of carnation， and the positive water balance value indicated that the cut flower absorbed more water than the water it lost. On the 2nd d， treatment T3 and the CK， T1， T4 and T2 all absorbed water， and compared with the 1st d， the carnation cut flower absorbed water continuously. On the 4th d， the water balance value of treatment T1 reached the maximum value， i.e.， the water absorbing capacity reached the maximum value， while the water absorbing capacity of the CK decreased continuously， and the cut flower in the CK began to wilt. A negative water balance value indicates the cut flower loses more water than the water it absorbs， so the flower will have a tendency of wilting. On the 4th d， the water absorbing capacity of treatment T3 reached its maximum value， the water absorbing capacity values of treatments T4 and T2 were also the largest than the values on the 0th d and the 2nd d， while the CK exhibited a negative value， indicating that the cut flower began to lose water. On the 6th d， the water absorbing capacity of the CK decreased， and the cut flower wilted， indicating that the CK suffered serious water loss. On the 8th d， treatment T1 began to absorb water slowly， and was measured with an error， while treatment T2 and treatments T3， T1 and T4 wilted slowly， and the CK was suffering increasingly serious water loss. Since the 10th d， the wilting of various treatments became more and more serious. The formula solution of treatment T1 was the one capable of prolonging the vase life of cut flower
Fig. 1 Effects of different solutions on water balance value of carnation cut flower
Fig. 2 Variation of relative fresh weight of carnation cut flower under different treatments among all the treatments. It could be seen from the figure that 6BA and B9 could remarkably improve the water condition in flower body， and retarded the wilting process caused by water loss.
Variation of relative fresh weight of cut flower under different treatments
For the effects of different preservative solutions on the relative fresh weight of carnation cut flower （Fig. 2）， within certain culture time， the relative fresh weights of treatments T1， T2， T3 and T4 were higher than that of the CK， and from the 0th d to the 4th d， under the different preservative treatments， the relative fresh weight of the carnation cut flower increased continuously， i.e.， the water absorption of carnation was promoted. The relative fresh weight of treatment T1 increased from the beginning of the experiment， and decreased from the 8th d. The relative fresh weight of treatment T2 began to decrease on the 8th d， and the carnation wilted， indicating that its vase life was going to be ended. The relative fresh weight of treatment T3 began to decrease on the 8th d， indicating that the carnation cut flower began to wilt， the vase life was going to be ended. However， the relative fresh weight of the CK decreased continuously， and the vase life was also on the decrease. Treatment T4 exhibited a decreasing trend from the 4th d.
Discussion
In this study， B9 is a kind of antiaging growth regulator， which has bactericidal effect and is applied widely. It could serve as dwarfing agent， fruit setting agent， rooting agent and preservative， and could simultaneously delay the opening of flower buds and retard aging. It could slow down the growth of flower after collection to a certain degree， prevent the biosynthesis of gibberellic acid in tissue and its metabolic process through the inhibition of plant growth， and improve the tolerance of cut flower against stress， thereby prolonging the vase life of cut flower. 6BA could prolong the vase life of carnation cut flower by affecting the water absorption of carnation cut flower and reducing respiratory rate. Zheng et al.[4] studied the physiological effect of 6BA and kinetin against the aging of carnation cut flower. In the study， 3% surcose+200 mg/L 8hydroxyquinoline + 200 mg/L citric acid +50 mg/L 6BA could prolong the vase life of cut flower to 23.5 d， while 3% sucrose + 50 mg/L citric acid +600 mg/L vitamin C +20 mg/L 6BA in this study could prolong the vase life of carnation to 16.4 d， which is shorter than that in previous studies. Therefore， 8hydroxyquinoline could remarkably prolong the vase life of cut flower， and the solution formula in the experiment still should be improved. Zeng et al.[8] studied the freshkeeping effect of the combined application of CaCl2 and B9 on carnation cut flower， and the combination of 250 mg/L B9 and 0.1 % CaCl2 exhibited the best freshkeeping effect， under which the vase life and fresh degree of carnation were the best. In the study， treatment T2 used 200 mg/L B9， with which the flower diameter and crown height of the cut flower increased remarkably， so the B9 concentration in the solution was proper. Yuan et al.[7] investigated the freshkeeping effect of environmentfriendly freshkeeping solution on carnation cut flower. The results showed that 7.5% sucrose +2.5 mg/L citric acid +2.5 mg/L 8HQ had the best treatment effect. The solution had remarkably antiaging and freshkeeping effects on carnation cut flower. Specifically， it could remarkably prolong the vase life of carnation cut flower and increase the water curing time by 20 d. This formula prolonged the vase life of carnation cut flower， but the fresh weight had no remarkable change. The comparison of this study with previous studies indicates further study is still needed， so as to find a formula which not only could prolong the vase life of cut flower， but also could improve the fresh degree of cut flower. Conclusion
In this study， the four pretreatment solutions could prolong the vase life of carnation cut flower to different degrees， and also could achieve of the effects of increasing flower fresh weight and flower diameter as well as crown height， promoting flowering and keeping water balance. Among all the treatments， treatment T1， 3% sucrose + 50 mg/L citric acid +600 mg/L vitamin C+20 mg/L 6BA had the best effect， and the vase life of carnation cut flower reached its maximum value of 16.4 d. Treatment T1 is a preferable formula with good stability and good safety， which could remarkably improve economic and ornamental value of carnation cut flower.
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