Growth, Flowering and Physiological Characteristics of Phalaenopsis affected by Nutritional and Environmental Factors

Phalaenopsis has established itself as the most valuable potted plants (18 million pots in sold in 2003, compared to mere 50,000 plants in 1983 in Dutch flower auctions Lopez and Runkle, 2005). In the USA Phalaenopsis alone tops with 70 - 90 of the entire Orchid group (Griesbach, 2002 Nash, 2003). Aesthetic beauty appeal, possibly olfactory attractions and the distinctly unique shelf life of these flowers even for as much as 4 long months and in a very wide spectrum of floral colors and color combinations are some of the suggested reasons. Increasing trends of Phalaenopsis domination is likely to be sustained in future if not going down.

Many market oriented breedersresearches have examined several external and recently even internal genetic factors for maximizing the flower production in Phalaenopsis in faster times because orchids in general require 4-6 years of juvenile phase from the seed stage to the stage of maturity to flower (Goh and Arditti, 2005), but most commercial orchids take only about 2-3 years probably because they are mostly tissue culture propagated. Environmental and nutrient factors then become critical for induction of flowering and therefore appropriate conditions have to be provided. This paper examines these issues.

In general there is an ongoing discussion regarding the best relative proportions of vegetative growth in relation to maximum flowering responses particularly in floriculture crops. There is a consensus that plant has to be culture in the best environment with most effective nutrient supply for the best consequences flowering. With over about 50 or so species in the genus Phalaenopsis (Pridgeon, 2000 cited by Lopez and Runkle, 2005)  and the ease of creating new hybrids coupled with ultra-rapid large scale in vitro micropropagation of  the selected hybrids provide a comfortable situation for rearing of plants. But the species diversity in floral characters such as the color combination cut flower shelf life, length of the spike itself and also more importantly the environmental and nutrient factors required for good vegetative growth just adequate for best flowering responses are quite variable. These and their combinations prevent any generalized scheme of adjusting these for maximum benefits. Further, results so far do not provide a comprehensive information on the interactions of the two broad factors and majority of studies have been confined only either to vegetative (juvenile) stage specific to different popular genera or hybrids, or to early and pre-flowering phase of this genus.

CAM in Phalaenopsis
Phalaenopsis is an epiphytic orchid characterized by Crassulacean Acid Metabolism. After the first comprehensive review on Crassulacean Acid Metabolism (CAM) was published by Wolf in 1960), many extensive researches have been carried out and reviewed periodically (Lttge, 2004). But in most of these orchids in general and Phalaenopsis in particular have been generally ignored. Lttge (2004) describes CAM as given by Osmond (1978) characterized by nocturnal uptake of CO2 via open stomata, fixation by phosphoenolpyruvate carboxylase (PEPC) and vacuolar storage of CO2 in the form of organic acids, mainly malic acid (phase I  HYPERLINK httpaob.oxfordjournals.orgcgicontentfull936629 l MCH087C205 Osmond, 1978), and (2) daytime remobilization of vacuolar organic acids, decarboxylation and refixation plus assimilation of CO2 behind closed stomata in the Calvin-cycle (phase III) .

Crassulacean Acid Metabolism (CAM) is an ecophysiological modification of photosynthetic carbon acquisition as a deviation from the well known C3 and C4 Plants. In all of these types carbon fixation pathways, six major environmental parameters CO2, H2O, light, temperature, nutrients and salinity participate in a coordinated manner at the whole plant level and also as affected by some or all these parameters. Each of these is significant in their different qualitative forms such as diurnal variations in temperature, light duration, quality and radiance and so on. 57 of all epiphytes are CAM plants.

Water use efficiency (WUE) might be the major ecophysiological property of CAM. CAM is a CO2-concentrating mechanism for adaptation to water stress because stomatal water loss through open stomata during dark is reduced by CO2 mobilization fixation (Luttge, 2004). In a CAM plant water is the most important factor as an adaptation to water-stress. Epiphytic species are especially more sensitive water stresses, in addition to particular problems of other types of plants. In a CAM plant water is the most important factor as an adaptation to water- stress because moisture loss through leaf stomata is reduced by CO2 absorption during dark via open stomata and subsequent fixation when stomata close during day time.

That Phalaenopsis is an obligate type of CAM plant is strengthened further by the experiments of Chen et al. (2008). They showed that the two groups of mature Phalaenopsis aphrodite subsp. formosana plants showed a clear diurnal oscillation of stomatal conductance, net CO2 uptake rate, malate and starch levels, and the phosphoenolpyruvate carboxylase and NAD-malic enzyme activities.

Growth and flowering have been generally excluded from each other. A discussion of environmental factors has to consider various parameters necessary for both roots zone and shoot region. Humidity and moisture, temperatures during days and night and their relations, photoperiod lengths, light intensity and quality are the major factors for both the roots and also the leaves and shoots.  

Root Zone, Potting Media, Water levels and Electrical Conductivity
Moisture in the root zone is particularly important. Humidity in the shoot zone is to be maintained around 60 - 70 and has to be critically monitored especially because Phalaenopsis is a monopodial kind of orchid. It may suffice to mention here that Wang et al. (2007) stress that inducing Phalaenopsis into flowering become easy if a good and a healthy root system is ensured. They suggest an ideal composition of a useful medium and efficient watering and fertilizer management in the early stages. Watering is crucial as Phalaenopsis is succulent unlike others which have pseudo-bulbs such as Cattleyas. Watering should be done more carefully in Phalaenopsis because of its succulent leaves. Roots should be allowed to nearly dry before rewatering can be started But can tolerate water deficiency for long periods but not more than two weeks. Also, in prolonged water deficient situation leaf photosynthetic carbon dioxide fixation goes down gradually. Long roots with light greenish tips indicate active plants.  

In the early studies such as by Sheehan (1960) examined two potting media, a specialty of orchids. This was among the first few papers which emphasized the need for well designed experiments to produce consistent results rather than just report only observations. These results also point out the importance of two commercial bark samples used due to their structure and differentially abilities to hold or release moisture and nutrients for availability to the roots.

Doritaenopsis cv. Labios and White Castle were grown with three different growing media Perlite, perlite  coir dust (31 vv) and perlite  rockwool granule (31 vv). Regardless of cultivars, plant growth on perlite  rockwool granule (31) was greatest. Perlite  rockwool granules had the best combinations of key physical characteristics such as total porosity and bulk density in addition to desirable combinations of high moisture capacity and container capacity with good air space, and electrical conductivity (Table 1). This was further proven by growing the Phalaenopsis plantlets (Table 2). Leaf pigments, chlorophylls and carotenoids were maximum on sphagnum moss followed by peat moss  perlite. Phalaenopsis plants were grown on this potting medium with three levels of media moisture content (50, 70 and 90 maximum water holding capacity) were examined. Nutrient solution with EC 1.0 dSm-1 was supplied during the culture. Plants grown at 90 showed highest growth rate but N, P, K, Ca and Mg contents in leaves and roots were fluctuating indicating that the growth rate was not related to these.

Table 1 Physical and chemical characteristics of growing media used in the experiment
         
Parameter      
MediaPerlitePerlite  coir dust Perlite  Rockwool granules (31, vv)
Total porosityz70.675.278.6
Container capacity y35.348.245.0
Air space ()35.227.032.6
Moisture capacity64.473.369.2
Bulk density (g.ml -10.180.180.16EC (dS.m-10.050.120.08Ph7.785.248.87z  
Percentage volume in container
y Predicted percentage volume at drainage

Table 2 Effect of growing medium on chlorophyll content in leaves of Doritaenopsis Tinny Tender after nine months of culture

Growing
mediumChlorophyll content (g. g -1FW)Chlorophyll abCarotenoids (g. g -1FW)   a     b A  bCoconut chips260.8 bz89.4 b350.2 b2.9 a85.7 bBark273.8 ab98.2 b372.0 ab2.8 b96.9 abPeat moss  perlite286.3 ab103.6 b389.9 ab 2.7 b93.3 abSphagnum moss297.5 a125.6 a423.1 a2.4 c101.9 az Mean separation within columns by Duncans multiple range test,   0.05

When EC of the nutrient solution maintained at 0.8 dSm-1, this potting medium resulted in the highest growth with higher number of roots than others. N, P, K, Ca and Mg contents in leaves and roots were also higher in plants grown on perlite  rockwool granule (31) than in perlite and perlite  coir dust (31).

Among four different growing media coconut husk, bark, peat moss-perlite (11) and sphagnum moss, fresh and dry weights of the White - flowered Doritaenopsis Tinny Tender plants grown in peat moss  perlite (11) mix and sphagnum moss were more than those in bark and coconut chip media. The same media resulted in better leaf growth, chlorophyll content, and N and P levels.  These media however showed lower Ca and Mg contents than in bark and coconut chips. The same two media also resulted in more flowers per plant, longer inflorescences, higher number of flowers per inflorescence, larger diameter of flowers.  Inflorescence emergence was unaffected by growing medium. Similar results were observed in another cv Labios

Doritaenopsis plants planted in culture bed filled with perlite  rock wool granule (31, vv) mix and 4 EC levels of nutrient solution were supplied. The nutrient solution for the experiment was developed by Chungbuk National University (CBNU-Phl2). At an early stage, in both cultivars, leaf growth increased with increasing EC level, of the nutrient solution, while root growth decreased with increasing EC levels. At the end of the experiment, plants grown on EC 2.0 dSm-1 showed lowest growth rate, while EC 0.8 dSm-1 resulted in the greatest plant growth. N, P, K and Mg contents in leaves increased with increasing levels of the nutrient solution but Ca contents decreased. During the first flowering season, in both cultivars, EC 1.6 dSm-1 induced larger number and size of flowers and longer stalks compared to other EC levels. Flowering was delayed by increasing EC level. During the second and third flowering season, higher flower quality was obtained at EC 0.8 dSm-1 in both cultivars. Doritaenopsis cv. Labios and White Castle were grown with three different growing media Perlite, perlite  coir dust (31 vv) and perlite  rockwool granule (31 vv). Regardless of cultivars, plant growth in perlite  rockwool granule (31) was greatest. EC of the nutrient solution maintained at 0.8 dSm-1 resulted in the highest growth with higher number of roots than others. N, P, K, Ca and Mg contents in leaves and roots were also higher in perlite  rock wool granule (31) than in perlite and perlite  coir dust (31).

Inflorescence length, number of flowers per inflorescence and flower size were greatest in perlite  rockwool granule (31) medium but inflorescence emergence was unaffected by the growing media.

Phalaenopsis plants were grown in perlite  rockwool granule (31 vv) and three levels of media moisture content (50, 70 and 90 maximum water holding capacity) were examined. Nutrient solution with EC 1.0 dSm-1 was supplied during the culture. Plants grown at 90 showed highest growth rate. On the other hand, N, P, K, Ca and Mg contents in leaves and roots were not related to the growth rate, showing fluctuations in each element. The relative water content (RWC) increased with increasing media moisture content. Chlorophyll a, chlorophyll b, and total chlorophyll content (fresh weight mgg) were highest at 90 media moisture content. Net CO2 uptake, stomatal conductance and transpiration in leaves during dark period increased progressively with increasing media moisture content, which became more significant with the duration of experimental period.

Effects of Environmental Factors and Composition of Nutrient Solution

Drought
Five-month-old in vitro cultured Doritaenopsis orchids transplanted and maintained under ex vitro condition in a greenhouse for 8 months were supplied with four nutrient solutions (at 12S, 1S, 32S and 2S with electrical conductivities of 0.9, 1.4, 1.9 and 2.4 dSm1, respectively) in ebb and flood irrigation system. Drought was imposed by withholding the supply of nutrient solution. These plants were analyzed for photosynthesis, stomatal conductance, transpiration, reducing sugar, total sugar, starch, protein, phosphoenolpyruvate carboxylase, superoxide dismutase, and peroxidase activity

Under light and dark condition photosynthesis, stomatal conductance and transpiration decreased with increasing the duration of drought. With the imposition of drought reducing sugar content increased while total sugar and starch content decreased. Protein content increased at 12S nutrient solution while decreased maximally at 32S. Drought also caused increase in phosphoenolpyruvate carboxylase and peroxidase activities but reduced superoxide dismutase activity.  Rewatering after 15 days of drought resulted in sudden revival from the stress related effects.

The relative water content (RWC) increased with increasing media moisture content. Chlorophyll a, chlorophyll b, and total chlorophyll content (fresh weight mgg) were highest at 90 media moisture content. Net CO2 uptake, stomatal conductance and transpiration in leaves during dark period increased progressively with increasing media moisture content, which became more significant with the duration of experimental period. On perlite  rockwool (31) medium, while spike emergence was unaffected, inflorescence length, number of flowers per inflorescence and flower size were greatest.

Doritaenopsis plants planted in culture bed filled with perlite  rockwool granule (31, vv) mix were fed with 4 EC levels of nutrient solution supplied for the experiment and developed by Chungbuk National University (CBNU-Phl2). At an early stage, in both cultivars, leaf growth increased with increasing EC level, of the nutrient solution, while root growth decreased with increasing EC levels.

Pre-flowering Vegetative Stem Growth
The vigor of a plant is generally reflected in its response in flowering. The vigor of a plant is generally reflected in its response in flowering. Cool night temperatures promote spike formation (Chen et al., 2008). Under constant prolonged duration of a high day and night temperature of 280C failed to induceproduce the flowering spikes but cooler temperature for at least 12 hoursday for about 6-8 weeks promoted inflorescence development in P. amabilis. This confirms Wents earlier report of observing more flower formation during cooler (20 C) at short day than at long day photo-periods. Similar observations were reported by Yoneda et al. (1991). Conversely, a diurnal fluctuation of high day and low night temperature promoted spike induction of Phalaenopsis (Lee and Lin 1984). Going by analogy provided by Sudoi and Tsutsui (1978), during night carbon dioxide fixation is more and reduces with the onset of the sunrise. In these reports, cooler temperature also stimulates flower initiation at cooler temperatures. Therefore if cooler temperatures promote greater Carbon fixation during it is very likely the spike initiation is the result of this greater carbon fixation. Sakanishi et al (1980) suggest that short day conditions may supplement to the positive lower temperature effects on spike formation and flower formation.  

Effects of Environmental factors on Physiological Characteristics of Doritaenopsis
Development of photosynthetic properties are very important for in vitro plantlets, which could be improved by altering their environmental growth conditions, such as increasing light intensity, humidity, air temperature or CO2 concentration. Humidity and temperature are crucial not only for ex vitro acclimatization of plantlets but also for a very rapid development of all normal physiological functions in a high value ornamental crop such as Phalaenopsis.

In vitro propagated Doritaenopsis New Candy plantlets acclimatized for one month. They were treated with three humidity levels (50, 70 and 90 251 C, at 12 h12 h photoperiod was used under fluorescent light (10010_molm2 s1 PPFD). In another experiment plantlets were maintained at five different temperatures 201, 251, 351 and 351 C, under 10010_molm2 s1 (PPFD). They were analyzed for different physiological characteristics including CO2 assimilation (An), stomatal conductance (Sc), transpiration rate (Tr), chlorophyll content, fresh and dry weight, leaf length, leaf area, leaf width, formation of new roots and survival rate.

Increasing humidity promoted plantlet growth as observed in fresh and dry weights, leaf dimensions, and survival rates but the number of new roots was not different. At 90 humidity fresh weight increased about 30 compared to 50 humidity.

RWC was reduced significantly after second day of transfer from in vitro to the growth chamber at 50 and 70 humidity compared to 90 humidity but as the acclimatization day progressed, RWC was completely recovered at the end of acclimatization period under all humidity stress.

Table 7 Leaf width, leaf length, leaf area of leaves, number of new roots, fresh weight, dry weight and survival rate of Doritaenopsis as affected by relative humidity (RH) and air Temperature after 30 days of acclimatizationx
RHair temperatureFresh weight (g)Dry weight (g)LeafNumber of new rootSurvival rate ()
Width (cm)Length (cm)Area (cm2Humidity506.63 b0.46 c4.0 a7.4 b9.17 b2.3 a62.2 b706.32 b0.51 b4.0 a7.7 b9.49 b2.3 a66.7 b909.46 a0.69 a4.3 a11.7 a11.17 a2.6 a92.2 aTemperature15 C    4.19 c0.30 c3.2 b6.0 b9.2 b1.0 b84.4 b
20 C7.48 ab0.54 a4.5 a6.6 a11.6 a2.3 ab91.1 a
25 C8.32 a0.59 a4.9 a6.9 a12.6 a3.0 a93.3 a
30 C6.42 b0.43 b5.1 a6.7 a12.7 a2.7 a92.2 a
35 C5.07 c0.36 c4.9 a6.6 a12.2 a2.8 a90.0 a

Mean separations within columns by different letters are significantly different according to Duncan s multiple range tests at 5 level x From Jeon et al., 2006

At 15 and 35 C, fresh weight decreased about 50 and 40, respectively compared to 25 C. Shoot dry weight decreased at both the low temperatures (at and below 20 C) and high temperature (35 C ) compared to 25 C. At low temperature (15 C), leaf area reduced more than 27 while it was less affected at high temperature (35 C) compared to 25 C grown plantlets (Table 7).

While at higher humidity (90), about 11 more new root was formed at 90 humidity compared to 50 humidity, higher temperature (25 C and above) promoted more root formation than at lower temperature (15 C).

In contrast, in temperature treated plantlets, dehydration became more serious at 15 C and RWC decreased after second day of transfer from in vitro to the growth chamber significantly and could not recover at the end of acclimatization period compared to other temperature (Fig. 2B). The dehydration was indicated by decrease of RWC.

Photosynthetic efficiency
During acclimatization on the second day itself, 50 and 70 humidity and low (15 C) and high temperature (30 and 35 C) FvFm was inhibited but recovered completely under all humidity at the end of acclimatization period (Fig. 3A). Similarly, at 15 C, FvFm decreased significantly and did not recover completely compared to 25 and 30 C grown plantlets.

Pigment content
Chlorophyll content was reduced at both low and high temperature compared to 25 C grown plantlets. About 12 chlorophyll a, 9 chlorophyll b and 12 total chlorophyll contents were decreased at 50 humidity compared to 90 humidity. At low temperature, 12 chlorophyll a, 17 chlorophyll b and 14 total chlorophyll and at high temperature 13 chlorophyll a, 11 chlorophyll b and 11 total chlorophyll were reduced compared to 25 C. The chlorophyll ab ratio was neither affected by humidity nor by temperature stress Jeon et al., 2006).

CO2 assimilation (An), stomatal conductance (Sc) and transpiration rate (Tr)
 
Under all humidity and temperatures conditions Doritaenopsis showed a typical CAM pattern of gas exchange. Both temperature and humidity stress, CO2 uptake (An) increased rapidly just after beginning of darkness and as it is a CAM orchid. An was found at night period only at both temperature and humidity stress. After 5 days of transfer, An, Sc, and Tr were decreased more at 50 humidity than 70 and 90 humidity (data not shown) and after 25 days of acclimatization, An, Sc, and Tr were increased even at 50humidity. In contrast, at 15 C, An, Sc, and Tr were highly reduced after 5 days of transfer (data not shown) and could not recover even after 25 days of acclimatization. However, under temperature, An, Sc, and Tr were higher at both 25 and 300 C compared to other temperatures. An, Sc, and Tr reached a maximum at 1900 and 0500 h under all humidity stress and all air temperatures treated plantlets. The most striking result of the present study is that increase in CO2 uptake was found in daytime at 150 C, which is also associated with the increase in stomatal conductance and transpiration except the remaining temperature.

Peroxidase activity (POD)
Peroxidase activity was determined after 30 days of acclimatization under both humidity and temperature in root. POD activity was not induced or inhibited under all humidity stresses (Fig. 2A) whereas at different temperature POD activity was highly induced and at 150 C POD activity was induced about 40 compared to 250 C (Fig.2B). However, at 20, 30 and 350 C, POD activity remained same but showed higher activity than at 250 C. Surprisingly, POD activity was not detected in leaves of Doritaenopsis (Jeon et. al., 2006).

Fig. 1. Diurnal changes in net CO2 assimilation (A), stomatal conductance (B) and transpiration (C) under humidity and net CO2 assimilation (D), stomatal conductance (E) and transpiration (F) under air temperature in Doritaenopsis grown in growth chamber.  Values are mean  S.E. (n  5).

Fig. 2 The peroxidase activity under humidity (A) and air temperature (B) stressed Doritaenopsis grown in growth chamber. Different letters are significantly different between the treatments at 5 level according to DMRT. Values are mean  S.E. (n  3).
x From Jeon et al., 2006

Effect of Nutritional and Environmental Factors on Flowering
The popularity of Phalaenopsis is just due to its attractive flowersinflorescences with many colorscolor combinations, shapes, scent, and prolonged cut flower shelf life only for its flowers. Interest in cultivating Phalaenopsis is justified and critical care has to be taken to provide appropriate environmental as well as nutritional conditions for promoting flowers in large marketable numbers in short time. Marketability is important because Blanchard et al (2007) observed that only about 50 of the flowers produced really have good market value.

Only recently there has been some activity in working out the necessary parameters accurately for producing repeatable results from a growers point. Until recently it was just accepted that temperatures higher than 25 C inhibited reproductive growth and lower temperatures than 25 C promote flowering (Ichihashi, 2003). Ichihashi studied light, carbon-nitrogen balance and cool temperatures and still concluded that more studies were required.

Jeon et al (2006) studied carbon metabolism in relation to spiking. Cool night temperatures promoted accumulation of free sugars. cool night temperatures (15  20 C ) also promote flowering  and also that temperature differences in night and day were critical (Chen et al.,  2008).

Recently Newton et al., (2009) investigated six different varieties of Phalaenopsis and found that there were cultivar specific differences with respect to exposure to 12 hours and longer at higher temperatures but still for majority of the varieties, cool temperature for less than nine hours seemed necessary for flower induction and formation. Earlier Sakanishi et al (1980) had reported that a short day condition may add to low temperature effects on flower induction.

Higher nitrogen levels promoted longer flower stalk length, inflorescence length and number of flowers per stem than lower nitrogen levels (Ruamrungsri et al., 2007).

Conclusions
Orchids are extremely in species diversity with genomic flux and still evolving. Many intergeneric hybrids are still possible. Orchids have a genome which is considered the shored of all. Angiosperms (Luttge, 2004). Yet number of varieties released by breeders is very high
In such a situation, it may not be out of context to mention that a generalized mechanism of cultural conditions for best and consensus responses from Phalaenopsis cultivars may be unexpected. It is worth mentioning that the kind of CAM plasticity observed in different eighteen individual species across Cymbidium reported recently by Motomura et al. (2008) due to weak andor strong CAM habits related to their respective habitats may exist in Phalaenopsis.

It is just the beginning of investigating into physiological and ecophysiological factors needed for this crop in general and for all its new and old popular varieties. Even with the limited amount of information, it is to be noted that there are possible and observed variations in responses to the environmental factors and nutritional aspects investigated so far. A lot more of information would be necessary to even hypothesize a theoretical scheme of factors for optimum productivity in Phalaenopsis plants and inflorescences. There is some new information on molecular aspects of some functional genes   including flowering phenomena. With the new and latest tools breeding for trait related factors may become easier.

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