PHYSIOLOGICAL CONTROL OF CHAMISE SHOOT GROWTH AFTER FIRE

Five controlled burns (ca. 2 ha each) were conducted in the Coast Range of northern California near Hopland, California, between November 18, 1975 and September 15, 1976 to determine the effect of shrub phenology on the sprouting response of chamise (Adenostoma fasciculatum H&A) following fire. The time of burn had little effect on the amount of shoot growth that occurred after fire, although the pattern of growth was altered. Shrubs burned on June 22 or August 2, 1976 grew continually until August 1977, while unburned shrubs or those burned at other times ceased seasonal growth during the first summer after fire. Neither the amount nor the pattern of shoot growth was influenced by the shrub water status before the fire occurred. The relationship between chamise shoot growth after fire, ¹⁴CO₂ fixation, xylem sap tension, and air temperature was also studied. The growth rate of regrowing shrubs was highly correlated with ¹⁴CO₂ fixation prior to the growth measurement. Water status (pre-dawn xylem sap tension) had a negative correlation with ¹⁴CO₂ fixation. The abundance of carbohydrate at the shoot apex is believed to influence the seasonal pattern and rate of chamise shoot growth following fire.     

Cryopreservation of in vitro-grown shoot tips of 'Troyer' citrange [Poncirus trifoliata (L.) Raf. × Citrus sinensis (L.) Osbeck.] by encapsulation-dehydration

. In vitro-grown shoot tips excised from preconditioned stock shoots of 'Troyer' citrange were successfully cryopreserved by encapsulation-dehydration. Optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 17.1% water content. The sucrose concentration in the preconditioning medium significantly influenced the growth and dry matter percentage of the stock shoots as well as subsequent survival of the cryopreserved shoot tips. Maximal growth of stock shoots was obtained in sucrose concentrations in the range of 0.15 M to 0.29 M, while the dry matter percentage increased as sucrose concentration increased up to 0.44 M. The survival of cryopreserved shoot tips increased from 40% to approximately 80% as the sucrose concentration for stock shoots increased from 0.09 M to 0.22 M or 0.29 M. The benzyladenine concentration in the post-culture medium significantly affected the survival and regrowth of the cryopreserved shoot tips. Survival of the shoot tips was lowest when they were post-cultured on benzyladenine-free medium. However, high benzyladenine concentrations (3-4 µM) induced callus formation. Optimal recovery was obtained in post-culture medium containing 2 µM benzyladenine and 0.05 µM !-naphthalene acetic acid. The extraction of shoot tips from alginate beads greatly improved the regrowth of cryopreserved shoot tips.     

EFFECT OF GROWTH RATE,MORPHOGENIC ACTIVITY,AND PHYLOGENY ON SHOOT APICAL ULTRASTRUCTURE IN OPUNTIA POLYACANTHA (CACTACEAE)

Dormant short shoot apices of Opuntia polyacantha were cultured under three conditions: cytokinin and high sucrose to stimulate the formation and rapid growth of a leafy long shoot; cytokinin and no sucrose (slow growth of a leafy long shoot); gibberellic acid and high sucrose (rapid growth of a spiny short shoot). These meristems, and also dormant (uncultured) ones, were analyzed by stereological, ultrastructural techniques. By comparing meristems growing with cytokinin but with or without sucrose, correlations between metabolic rate and apical ultrastructure were studied; comparison of leaf-producing and spine-producing meristems permitted examination of correlations with morphogenic role; comparison with published data for four other species permitted study of phylogenetic effects, and comparison with dormant apices revealed information about meristem activation. Ultrastructure varied according to each condition: metabolic rate, morphogenic activity and species can be distinguished by quantitative methods. Apical ultrastructure is most strongly correlated with rate of growth such that apices of differing species resemble each other if growing at similar rates, whereas apices of a single species differ markedly if growing at differing rates or if performing different morphogenic activities. Hyaloplasm is an excellent indicator of metabolic rate; mitochondria, nuclei, and vacuoles are not.     

Synergistic action of auxin and cytokinin mediates aluminum‐induced root growth inhibition in Arabidopsis

Auxin acts synergistically with cytokinin to control the shoot stem‐cell niche, while both hormones act antagonistically to maintain the root meristem. In aluminum (Al) stress‐induced root growth inhibition, auxin plays an important role. However, the role of cytokinin in this process is not well understood. In this study, we show that cytokinin enhances root growth inhibition under stress by mediating Al‐induced auxin signaling. Al stress triggers a local cytokinin response in the root‐apex transition zone (TZ) that depends on IPTs, which encode adenosine phosphate isopentenyltransferases and regulate cytokinin biosynthesis. IPTs are up‐regulated specifically in the root‐apex TZ in response to Al stress and promote local cytokinin biosynthesis and inhibition of root growth. The process of root growth inhibition is also controlled by ethylene signaling which acts upstream of auxin. In summary, different from the situation in the root meristem, auxin acts with cytokinin in a synergistic way to mediate aluminum‐induced root growth inhibition in Arabidopsis.     

WATER RELATIONS OF TWO CALIFORNIA CHAPARRAL SHRUBS

Water use patterns of two California chaparral shrub species, chamise (Adenostoma fasciculatum H. and A.) and Stanford manzanita (Arctostaphylos stanfordiana Parry), were compared during summer drought. Observations of diurnal and seasonal courses of shoot water potential, leaf conductance and transpiration revealed that chamise was more conservative in water use than manzanita. Evidence obtained cast doubt on a hypothesis previously proposed to explain an anomalous pattern of shoot water potential in chamise.     

Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro

Efficacy of phloroglucinol in promoting growth and development of in vitro-derived shoot tips was studied in six potato (Solanum tuberosum L.) genotypes. Different concentrations of phloroglucinol (0, 0.08, 0.4, 0.8, 1.2 and 1.6 mM) were tested in combination with either 0.1 or 0.2 M sucrose in shoot tip proliferation medium based on MS (Murashige and Skoog, 1962) medium supplemented with 5.8 μM GA₃ (gibberellic acid), 1.1 μM BA (N⁶-benzyladenine) and 8.39 μM D-calcium pantothenate. Phloroglucinol fostered multiple shoot formation, promoted axillary shoot proliferation in terms of shoot tip fresh weight and shoot length, and stimulated root formation on the shoot tips. There was significant phloroglucinol × sucrose interaction for number of shoots developed per shoot tip, shoot tip fresh weight and number of roots induced per shoot tip. The beneficial effect of phloroglucinol on shoot tip survival was conspicuous only in genotypes that showed poor survival in the control proliferation medium. There were significant differences in response between the two sucrose levels with regard to shoot tip fresh weight and number of roots per shoot tip. Phloroglucinol in combination with 0.2 M sucrose induced maximum number of roots per shoot tip. Optimum shoot tip growth was fostered in medium containing 0.8 mM phloroglucinol and 0.2 M sucrose. High frequency multiple shoot formation in this medium ensures a faster rate of potato shoot tip multiplication within a limited time and space. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of drought on sorbitol and sucrose metabolism in sinks and sources of peach

In peach (Prunus persica [L.] Batsch.), sorbitol and sucrose are the two main forms of photosynthetic and translocated carbon and may have different functions depending on the organ of utilization and its developmental stage. The role and interaction of sorbitol and sucrose metabolism was studied in mature leaves (source) and shoot tips (sinks) of ‘Nemaguard’ peach under drought stress. Plants were irrigated daily at rates of 100, 67, and 33% of evapotranspiration (ET). The relative elongation rate (RER) of growing shoots was measured daily. In mature leaves, water potential (Ψ_w), osmotic potential (Ψ_s), sorbitol‐6‐phosphate dehydrogenase (S6PDH, EC 1.1.1.200), and sucrose‐phosphate synthase (SPS, EC 2.4.1.14) activities were measured weekly. Measurements of Ψ_s, sorbitol dehydrogenase (SDH, 1.1.1.14), sucrose synthase (SS, EC 2.4.1.13), acid invertase (AI, EC 3.2.1.26), and neutral invertase (NI, EC 3.2.1.27) activities were taken weekly in shoot tips. Drought stress reduced RER and Ψ_w of plants in proportion to water supply. Osmotic adjustment was detected by the second week of treatment in mature leaves and by the third week in shoot tips. Both SDH and S6PDH activities were reduced by drought stress within 4 days of treatment and positively correlated with overall Ψ_w levels. However, only SDH activity was correlated with Ψ_s. Among the sucrose enzymes, only SS was affected by drought, being reduced after 3 weeks. Sorbitol accumulation in both mature leaves and shoot tips of stressed plants was observed starting from the second week of treatment and reached up to 80% of total solutes involved in osmotic adjustment. Sucrose content was up to 8‐fold lower than sorbitol content and accumulated only occasionally. We conclude that a loss of SDH activity in sinks leads to osmotic adjustment via sorbitol accumulation in peach. We propose an adaptive role of sorbitol metabolism versus a maintenance role of sucrose metabolism in peach under drought stress.     

Cryopreservation of African violet (Saintpaulia ionantha Wendl.) shoot tips

Summary Cryopreservation of African violet via encapsulation-dehydration, vitrification, and encapsulation-vitrification of shoot tips was evaluated. Encapsulation-dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2M glycerol plus 0.4M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5M sucrose or 5% DMSO plus 0.75M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2M glycerol plus 0.4M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation-vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.     

Cryopreservation of sour orange (Citrus aurantium L.) shoot tips

Summary The objective of this study was to establish a cryopreservation protocol for sour orange (Citrus aurantium L.). Cryopreservation was carried out via encapsulation-dehydration, vitrification, and encapsulation-vitrification on shoot tips excised from in vitro cultures. Results indicated that a maximum of 83% survival and 47% regrowth of encapsulated-dehydrated and cryopreserved shoot tips was obtained with 0.5M sucrose in the preculture medium and further dehydration for 6 h to attain 18% moisture content. Dehydration of encapsulated shoot tips with silica gel for 2h resulted in 93% survival but only 37% regrowth of cryopreserved shoot tips. After preculturing with 0.5M sucrose, 80% of the vitrified cryopreserved shoots survived when 2M sucrose plus 10% dimethyl sulfoxide (DMSO) was used as a cryoprotectant for 20 min at 25°C. Survival and regrowth of vitrified cryopreserved shoot tips were 67% and 43%, respectively, when 0.4M sucrose plus 2M glycerol was used as a loading solution followed by application of 100% plant vitrification solution (PVS2) for 20 min. Increased duration of exposure to the loading solution up to 60 min increased survival (83%) and regrowth (47%) of cryopreserved shoot tips. With encapsulation-vitrification, dehydration with 100% PVS2 for 2 or 3 h at 0°C resulted in 50 or 57% survival and 30 or 40% regrowth, respectively, of cryopreserved shoot tips.     

Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips

Shoot tips excised from dormant axillary buds of persimmon (Diospyros kaki Thunb.) were cryopreserved by vitrification. These excised shoot tips were dehydrated in a highly concentrated vitrification solution for 20 min at 25°C and then plunged directly into liquid nitrogen. After rapid warming in water at 40°C, the shoot tips were rinsed in a 1.2 M sucrose solution for 20 min and then plated on a solidified culture medium. Successfully vitrified shoot tips resumed growth within 10 days of plating and developed shoots within 3 weeks without intermediary callus formation. This simple protocol was successfully applied to the 16 cultivars found in the temperate zone. The average rate of shoot formation was 89%. Even the subtropical species of Diospyros demonstrated a very high recovery growth when the shoot tips had been previously osmoprotected with a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min following sucrose preculture. Little or no contamination occurred in the cryopreserved shoot tips excised from sterilized winter axillary buds. Thus, this simple and reliable vitrification protocol using dormant shoot tips appears to be promising as a routine method for the long-term conservation of Diospyros germplasm of both temperate and subtropical origins.     

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.     

Adventitious shoot formation is not inherent to micropropagation of banana as it is in maize

Despite their similar morphology, banana and maize shoot tips responded strikingly different with respect to the in vitro formation of homogeneous multiple shoot clusters. While up to 50 small shoots per maize explant could be induced within 1 month, zero to one additional shoot formed starting from a banana shoot tip. Subsequently, banana shoot tips were subjected to different combinations of five cytokinins (0–100 μM) and five auxins (0–5 μM). The cytokinins thidiazuron and benzylaminopurine stimulated multiplication to a higher extent compared to zeatin, kinetin and isopentenyl adenine. The addition of indoleacetic acid, naphthalene acetic acid or indolebutyric acid to cytokinin containing medium did not affect the in vitro response. In contrast, 2,4-dichlorophenoxyacetic acid (1 and 5 μM) and a higher concentration of picloram (5 μM) had a detrimental effect on shoot formation and resulted in explant death and globule development. When small (0.1 cm) shoot tips were grown on cytokinin medium without an auxin source, the average number of shoots was generally two to three times lower compared to bigger (0.5 cm) shoot tips. Based on our experience in maize and this large-scale study with banana shoot tips, we conclude that banana is extremely recalcitrant towards adventitious shoot formation. This recalcitrance could not be overcome by any of the 173 different plant growth regulator combinations tested. In vitro multiplication of banana thus appears solely restricted to axillary shoot formation.     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

Cryopreservation of in vitro-grown shoot tips of grapevine by encapsulation-dehydration

In vitro-grown shoot tips of the LN33 hybrid (Vitis L.) and cv. Superior (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration. Encapsulated shoot tips were precultured stepwise on half-strength MS medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1.0 M for 4 days, with one day for each step. Following preculture, encapsulated shoot tips were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved shoot tips were post-cultured on a post-culture medium for survival. An optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 15.6 and 17.6% water content for the LN33 hybrid and cv. Superior, respectively. Comparison between the effects of dehydration with silica gel and by air drying on cryopreserved shoot tips, showed that survival was dependent on water content, not on dehydration method. The thawing method markedly affected survival of cryopreserved shoot tips, and thawing at 40 °C for 3 min was found best. No callus formation and fastest shoot elongation were obtained when cryopreserved shoot tips were post-cultured on the post-culture medium composed of half-strength MS supplemented with 1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. With these optimized parameters, 60 and 40% survival of cryopreserved shoot tips were obtained for the LN33 hybrid and cv. Superior, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.     

Cryopreservation of apple (Malus×domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification

Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips (83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar. Received: 29 July 1999 / Revision received: 24 September 1999 / Accepted: 26 November 1999     

Plasmolysis and recovery of different cell types in cryoprotected shoot tips of Mentha × piperita

Summary. Successful cryopreservation of plant shoot tips is dependent upon effective desiccation through osmotic or physical processes. Microscopy techniques were used to determine the extent of cellular damage and plasmolysis that occurs in peppermint (Mentha × piperita) shoot tips during the process of cryopreservation, using the cryoprotectant plant vitrification solution 2 (PVS2) (30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, 0.4 M sucrose) prior to liquid-nitrogen exposure. The meristem cells were the smallest and least plasmolyzed cell type of the shoot tips, while the large, older leaf and lower cortex cells were the most damaged. When treated with cryoprotectant solutions, meristem cells exhibited concave plasmolysis, suggesting that this cell type has a highly viscous protoplasm, and protoplasts have many cell wall attachment sites. Shoot tip cells were most severely plasmolyzed after PVS2 treatment, liquid-nitrogen exposure, and warming in 1.2 M sucrose. Successful recovery may be dependent upon surviving the plasmolytic conditions induced by warming and diluting treated shoot tips in 1.2 M sucrose solutions. In peppermint shoot tips, clumps of young meristem or young leaf cells survive the cryopreservation process and regenerate plants containing many shoots. Cryoprotective treatments that favor survival of small, meristematic cells and young leaf cells are most likely to produce high survival rates after liquid-nitrogen exposure. Correspondence and reprints: National Center for Genetic Resources Preservation, U.S. Department of Agriculture, 1111 S. Mason Street, Fort Collins, CO 80521, U.S.A.     

Trehalose Toxicity in Cuscuta reflexa: SUCROSE CONTENT DECREASES IN SHOOT TIPS UPON TREHALOSE FEEDING

Trehalose, an α,α-diglucoside, induced a rapid blackening and death of shoot tips of Cuscuta reflexa (dodder) cultured in vitro. The onset of toxic symptom was delayed if any of the several sugars which support the in vitro growth of Cuscuta was supplied with trehalose. The rate of trehalose uptake or its accumulation in the tissue was not affected by sugar cofeeding. The levels of total and reducing sugars declined appreciably in the trehalose-fed shoot tip explants compared to control tissue cultured in absence of a carbon source. This was not due to an increased rate of respiration of the trehalose-treated tissue. In shoot tips cultured in presence of both trehalose and sucrose, the decline in total and reducing sugars was curtailed. There was a marked fall in the level of sucrose; and invertase activity was higher in trehalose-fed shoot tips. The incorporation of label from [¹⁴C]glucose into sucrose in the shoot tip explant was reduced as early as 12 h of trehalose feeding. The results suggest that increased utilization of sucrose as well as an inhibition of its synthesis contribute to the drastic fall in the sucrose content upon trehalose feeding.     

Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid

Cytokinin oxidase plays an important role in the cytokinin regulatory processes. We have cloned a novel putative cytokinin oxidase, DSCKX1 (D endrobium Sonia cytokinin oxidase), by mRNA differential display from shoot apices of Dendrobium Sonia cultured in the presence of BA. The DSCKX1 gene appears to have three alternative splicing forms and its expression of DSCKX1 was induced in a tissue-specific manner by cytokinins. In transgenic orchid plants overexpressing DSCKX1, the elevated level of cytokinin oxidase activity was accompanied by a reduction of cytokinin content. These plants exhibited slow shoot growth with numerous and long roots in vitro. Their calli also showed decreased capability of shoot formation. Conversly, antisense transgenic plants showed rapid proliferation of shoots and inhibition of root growth combined with a higher endogenous cytokinin content than wild-type plants. Thus DSCKX1 appears to play an important role on cytokinin metabolism and the related developmental programmes in orchid.