In vitro micropropagation of Basilicum polystachyon (L.) Moench and identification of endogenous auxin through HPLC

An efficient plant regeneration protocol was developed for Basilicum polystachyon (L.) Moench using shoot tip from in vitro germinated plant. Both shoot multiplication and root induction were initiated from shoot tip explants in Murashige and Skoog’s (MS) basal medium supplemented with N⁶-benzylaminopurine (BAP) and 6-furfurylaminopurine (Kin) combination with 1-naphthaleneacetic acid (NAA) and without any plant growth regulator. Among the different concentrations and combinations of growth regulators, the highest number of shoots per explants was induced on 13.32 μM BAP with 0.53 μM NAA. It was also found that the multiplication of shoots along with roots induced in MS medium without any plant growth regulators. The in vitro grown plants were successfully hardened and acclimatized in the field with a 99% survival rate. The results obtained from HPLC analysis established the presence of a significant amount of endogenous auxin, viz. indole-3-acetic acid acid and indole-3-butyric acid in the shoot and root tips of B. polystachyon. This is the first report of a successful multiplication of B. polystachyon in absence of plant growth regulators and the presence of an abundant quantity of endogenous auxin in root and shoot tips using Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) coupled with ultraviolet–visible (UV–Vis) detector.

     

Shoot tip culture and cryopreservation for eradication of Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from apple rootstocks ‘M9’ and ‘M26’

This study attempted to eradicate Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) from virus‐infected in vitro shoots of apple rootstocks ‘M9’ and ‘M26’ using shoot tip culture and cryopreservation. In shoot tip culture, shoot tips (0.2 mm in length) containing two leaf primordia failed to show shoot regrowth. Although shoot regrowth rate was the highest in the largest shoot tips (1.0 mm in length) containing four leaf primordia, none of the regenerated shoots was virus‐free. Shoot tips (0.5 mm in length) containing two and three leaf primordia produced 100% and 10% of ASPV‐free shoots, respectively, while those (1.0 mm) containing four leaf primordia were not able to eradicate ASPV. ASGV could not be eradicated by shoot tip culture, regardless of the size of the shoot tips tested. In cryopreservation, shoot tips (0.5 mm in length) containing two leaf primordia did not resume shoot growth. Although 1.0‐mm and 1.5‐mm shoot tips gave similarly high ASPV‐free frequencies, the latter had much higher shoot regrowth rate than the former. Very similar results of shoot regrowth and virus eradication by shoot tip culture and cryopreservation were observed in both ‘M9’ and ‘M26’. Histological observations showed that only cells in upper part of apical dome and in leaf primordia 1–3 survived, while other cells were damaged or killed, in shoot tips following cryopreservation. Virus immunolocalization found ASPV was not detected in upper part of apical dome and leaf primordia 1 and 2, but was present in lower part of apical dome, and in leaf primordium 4 and more developed tissues in all samples tested. ASPV was also detected in leaf primordium 3 in about 16.7% and 13.3% samples tested in ‘M9’ and ‘M26’. ASGV was observed in apical dome and leaf primordia 1–6, leaving only a few top layers of cells in apical dome free of the virus. Different abilities of ASPV and ASGV to invade leaf petioles and shoot tips were also noted.     

Independent control of organogenesis and shoot tip abortion are key factors to developmental plasticity in kiwifruit (Actinidia)

BACKGROUND AND AIMS: In kiwifruit (Actinidia), the number of nodes per shoot is highly variable and is influenced by genotype and environmental conditions. To understand this developmental plasticity, three key processes were studied: organogenesis by the shoot apical meristem during shoot growth; expansion of phytomers; and shoot tip abortion. METHODS: Studies were made of organogenesis and shoot tip abortion using light and scanning electron microscopy. The effect of temperature on shoot growth cessation was investigated using temperature indices over the budbreak period, and patterns of shoot tip abortion were quantified using stochastic modelling. KEY RESULTS: All growing buds began organogenesis before budbreak. During shoot development, the number of phytomers initiated by the shoot apical meristem is correlated with the number of expanding phytomers and the mean internode length. Shoot tip abortion is preceded by growth cessation and is not brought about by the death of the shoot apical meristem, but occurs by tissue necrosis in the sub-apical zone. For most genotypes studied, the probability of shoot tip abortion is higher during expansion of the preformed part of the shoot. Lower temperatures during early growth result in a higher probability of shoot tip abortion. CONCLUSIONS: Organogenesis and shoot tip abortion are controlled independently. All buds have the potential to become long shoots. Conditions that increase early growth rate postpone shoot tip abortion.     

Hormonal control of the outgrowth of axillary buds in <Emphasis Type="Italic">Alstroemeria</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

We study apical dominance in Alstroemeria, a plant with an architecture very different from the model species used in research on apical dominance. The standard explant was a rhizome with a tip and two vertically growing shoots from which the larger part had been excised leaving ca. 1 cm stem. The axillary buds that resumed growth were located at this 1-cm stem just above the rhizome. They were released by removal of the rhizome tip and the shoot tips. Replacement of excised tips by lanolin with indole-3-butyric acid (IBA) restored apical dominance. The auxin transport inhibitors 2,3,5-triiodobenzoic acid (TIBA) and N-1-napthylphthalamic acid (NPA) reduced apical dominance. 6-Benzylaminopurine (BAP) enhanced axillary bud outgrowth but the highest concentrations (> 9 μM) caused fasciation. Thidiazuron (TDZ) did not show improvement relative to BAP. Even though the architecture of Alstroemeria and the model species are very different, their hormonal mechanisms in apical dominance are for the greater part very similar.     

Relationship of Pruning and Growth Morphology with Hormone Ratios in Shoots of Pillar and Standard Peach Trees

Genotype and cultural management determine the shape of peach [Prunus persica (L.) Batch] tree canopies in orchards. Not well understood, however, is the relationship between terminal growth, lateral branching, and shoot hormone levels that can fundamentally affect tree canopy development. In this experiment, two peach cultivars with widely differing growth habits (Pillar, KV930479 and Standard, ‘Harrow Beauty’) were budded on ‘Lovell’ rootstock, planted in the field in 1998, and characterized for shoot morphology and hormone concentrations in 2002 and 2003 (the fourth and fifth leaf, respectively). Auxin (indole-3-acetic acid) and cytokinins (largely trans-zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) were measured in shoot tips (2002) and current-year shoots (2003) using mass spectrometry. In 2002, Pillar trees had less sylleptic branching, more upright growth, and higher auxin and auxin-to-cytokinin ratios than Standard trees. In Pillar trees in 2003, auxin concentrations and shoot growth were highest in current year shoots; in pruned trees, only auxin levels increased. Peach tree growth habits may be the result of altered hormone metabolism. Growth forms leading to superior production efficiency may be developed by selection based on specific target hormone concentrations and ratios.     

SHOOT TIP ABORTION IN ULMUS AMERICANA

Millington , W. F. (Marquette U., Milwaukee, Wis.) Shoot tip abortion in Ulmus americana. Amor. Jour. Bot. 50(4): 371–378. Illus. 1963.—Phenological observations of American elm have shown that the phenomenon of shoot tip abortion in which the distal several plastochrons of each shoot turn yellow and abort, inducing sympodial growth, occurs over a period of several weeks starting at the time fruits are shed from older trees. Time of abortion varies among plants of different age, among individuals of the same age, and among shoots on the same individual. In the latter case, time of abortion is inversely correlated with the vigor of the shoot, the most vigorous shoots on a branch being the last to abort. Abortion is first evident in the yellowing of the entire shoot tip. Cytohistological studies show that necrosis commences at the sixth to eighth node back of the apex, where it is apparent in autolysis of internal cells of the stipules. Necrosis progresses acropetally in the stipules and young leaves and ultimately involves the leaf primordia at the shoot apex. Mitosis ceases in the shoot apex and its meristematic appearance is lost. These changes follow in basipetal sequence in the axillary buds down to the bud below the abscission site. This bud remains active and will resume growth the following season. The abscission site is evident externally as a green-yellow boundary in the basal part of the internode. No protective layer is present at the time of abscission, but it develops after the shoot tip abscises. There is no indication of blocking of vascular tissues before shoot tip abortion and limitation of water supply probably is not a causal factor. Photoperiod studies show that shoot tip abortion is accelerated in short days and delayed but not prevented in long days. Greenhouse experiments show that abortion is delayed also in seedlings, in plants supplied with organic fertilizer, or grown with the roots unconfined. Plants grown in a nutrient solution deficient in nitrogen aborted ahead of controls and plants deficient in calcium. Although shoot tip abortion occurs coincident with fruit drop, there is no indication of a causal relationship. The literature relating to shoot tip abortion is discussed in relation to the above observations.     

Effect of Succinic Acid-2,2-Dimethylhydrazide on Endogenous Auxin Level in Apple Shoots

Endogenous auxin levels in the shoots of apple cv. Ingrid Marie (red mutant) treated with SADH (succinic acid-2,2-dimethylhydrazide) were measured. SADH was applied in aqueous solution at 12.5, 25 or 50 mM concentrations in June, 3 weeks after flowering. The defoliated shoot tips were analysed just before SADH spray and 7, 15, 30 and 60 days after spraying. Similar treatments had been made in earlier years. When analysed before the treatment of the year started, shoot extracts of control plants contained higher auxin level than those shoots which were to receive SADH treatment, indicating a residual effect of SADH treatmens during previous years. There was almost no auxin activity in the shoots analysed after 7, 15 and 30 days of SADH treatment. The control plants maintained a high level of auxin activity during this period. However, after 60 days of treatment, shoots treated with 25 or 50 mM exhibited higher auxin activity than control shoots. There appears to be no relationship between the SADH concentration and reduction in auxin activity in the shoots at early stages after treatment.     

EXPERIMENTAL CONTROL OF THE SHOOT SYSTEM IN SPORELINGS OF PTERIDIUM AQUILINUM

The shoot system of Pteridium develops from a simple, upright sporeling type through a rhizomatous leaf-bearing form, into a complex system with rapidly growing, leafless long shoots and slowly growing, leafy lateral branches or short shoots. Transitional stages axe available in sporeling material and ontogeny may be controlled by specific carbohydrate levels. At concentrations of sucrose below 2% a rhizomatous sporeling plant may be maintained indefinitely in the leaf-bearing state. At 2–8% sucrose, the short shoot habit becomes established, and at 4–8% it may be reliably stabilized. If concentrations up to 5 mg/liter of kinetin are added to cultures of intact plants or to excised short and long shoot apices of plants, the long shoots expand slowly into determined (no further growth possible even on transfer back to basal medium) club-shaped tips. The short shoot apices, by contrast, quickly proliferate into callus-like masses and will re-form organized meristems over the entire lobulate surface on transfer to medium lacking kinetin. The morphogenetic significance of these findings is discussed.     

Growth Regulators in Populus tremula IV. Apical Dominance and Suckering in Young Plants

Experiments with small plants of Populus tremula L. growing in solution culture indicate that polarly transported auxin is an important factor in the control of axillary bud growth. If the auxin supply from the growing apex is eliminated, the number of buds released is influenced by factors translocated in the transpiration stream from the roots. Suckers may be induced to develop from aspen roots, the age of which is six weeks or more. Removal of the growing apex and the axillary buds or stoppage of shoot growth by short day treatment were effective in inducing abundant suckering in small aspen plants. Some mature leaves had to be maintained, indicating the dependence of sucker formation on carbohydrate supply. These treatments are known to decrease auxin production in the shoots. Extraction and biological assay showed a decrease in the content of auxin in the roots as a consequence of removal of growing shoot parts. The results indicate that suckering in roots of intact aspen plants is prevented by auxin transported into the roots from growing shoot parts.     

An efficient in vitro plantlet regeneration of <Emphasis Type="Italic">Cryptocoryne wendtii</Emphasis> and <Emphasis Type="Italic">Cryptocoryne becketti</Emphasis> through shoot tip culture

An efficient micropropagation protocol was established for Cryptocoryne wendtii and Cryptocoryne becketti using shoot tips explants. Multiple shoots were induced from shoot tip explants of both species cultured on agar-gelled as well as liquid MS medium supplemented with 0.5 mg/L BA and 0.2 mg/L IBA (proliferation medium). The multiple shoots of both the species formed on agar-gelled as well as liquid medium were vigorously growing with well-developed roots and leaves after 4 weeks of culture. Highest number of multiple shoots was obtained from shoot tip explants of both the species cultured in liquid proliferation medium after 4 weeks of culture. The shoot tip explants of C. wendtii and C. becketti, that were cultured in liquid proliferation medium (2 weeks) followed by culturing on agar-gelled proliferation medium (2 weeks) also produced the multiple shoots. Shoot tips cultured on agar-gelled medium produced the least number of multiple shoots after 4 weeks of culture. Histological study did not show any abnormalities in the leaves of in vitro plantlets of both the species, cultured in agar-gelled and liquid proliferation medium. The leaves of the in vitro plantlets formed normal mesophyll cells and vascular bundles. More than 95% of the acclimatized plantlets grew vigorously without any morphological abnormalities.     

Apical Growth Cessation and Shoot Tip Abscission in Salix

Time course of apical shoot growth and shoot tip abortion in northern ecotypes (lat. 69°39′N, long. 18°37′E) of Salix pentandra and S. caprea have been investigated. In trees more than 15 years old growing under natural climatic conditions apical growth cessation and shoot tip abortion normally occurred in June-July when the day length still was 24 h. Application of GA₃, in spring to the apex effectively delayed growth cessation and shoot tip abortion. Application of kinetin was without effect. First-year seedlings of both species grew continuously at temperatue of 9 to 24°C in 24 h photoperiod. Short days induced apical growth cessation, but two to four (S. pentandra) or three to five (S. caprea) weeks of 12 h photoperiod were required to stop the elongation growth. The results indicated that the critical photoperiod for apical growth cessation in the used ecotype of S. pentandra was 16 to 18 h at 18°C. Short days had a minor effect only on the formation of apical leaf primordia in small seedlings. Development of axillary buds and radial growth were stimulated by short days when compared with long days. Small seedlings of both species (3 to 8 cm high at the start) formed terminal buds in short days, but in large seedlings (more than about 15 cm high) apical growth cessation was accompanied by shoot tip abortion. Abscisic acid applied to the apex or through a leaf did not induce growth cessation in S. pentandra seedlings grown in continuous light. The growth retardants CCC, B-9 and Phosphon D reduced growth rate under continuous light and induced shoot tip abortion in some plants. The effect of CCC was counteracted by GA₃. Apical growth cessation in short days was significantly delayed by a single GA₁ application.     

SHOOT TIP ABORTION AND SYMPODIAL BRANCH REORIENTATION IN BROWNEA ARIZA (LEGUMINOSAE)

The growth of the tropical tree Brownea ariza Benth. is modular and conforms to Troll's model. Distinctive anatomical features of its shoot development were investigated. Each module consists of from 6–10 compound leaves and terminates its growth by shoot tip abortion. Sympodial branch systems are formed by renewal growth from the most distal (pseudoterminal) 1–2 buds. New modules are wholly preformed within large (15–28 cm) buds. The flush occurs without a resting period and full shoot expansion is completed within one day. A distinct abscission zone develops in the stem just distal to the node of the last expanded leaf. Abortion of the shoot apex and 5–6 embryonic leaves occurs 2–3 days after the flush begins. This tissue vacuolates and begins to become necrotic prior to actual abscission. New flushes are pendent but are reoriented to a plagiotropic or upright position to create an arborescent form. Reorientation begins quickly (10° within 2 days) due to maturation of primary and secondary tissues and continues throughout the life of the branch by means of reaction wood formation on the upper surface.     

CONTROL OF SHOOT-RHIZOME DIMORPHISM IN THE WOODY MONOCOTYLEDON,CORDYLINE (AGAVACEAE)

In Cordyline terminalis negatively geotropic leafy shoots and positively geotropic rhizomes develop from single axillary buds on either shoots or rhizomes. All axillary buds have similar morphogenetic potential when released from apical dominance. Experiments in which the orientation of the apex is changed, organs removed, or growth regulators applied indicate that after a rhizome is initiated, it is maintained as a rhizome by auxin originating in the leafy shoot. When auxin levels are lowered by changes in the orientation of the axis or shoot removal, the rhizome apex becomes a shoot apex, which appears to be the stable state of the actively growing apex. Benzyl adenine when applied exogenously to the apex or lateral buds has the same effect as lowering the auxin level. Gibberellic acid has no effect on the apex or lateral buds. High levels of exogenous naphthaleneacetic acid cause bud release and development of rhizomes from previously inhibited axillary buds of the shoot. However, it was not possible to convert a shoot apex into a rhizome apex by auxin treatment. It is suggested that the release of buds on the lower side of horizontal branches and of buds directly above a stem girdle is caused by high auxin levels on the lower side or distal to the girdle. The experimental results are discussed in relation to naturally occurring shoot-rhizome dimorphism.     

Sympodial and monopodial branching inAcer (Aceraceae): Evolutionary trend and ecological implications

The evolutionary trend and its ecological implications in sympodial and monopodial branching patterns has been investigated in 20 JapaneseAcer spp. through comparison of shoot tip abortion and terminal bud formation. The genus is divided into two species groups according to its branching pattern, one (6 species) predominantly exhibiting sympodial branching with frequent monopodial branching in short shoots (sympodial species), and the other (14 species) exhibiting only monopodial branching (monopodial species). The early ontogeny of leaf and bud scales is described. Despite the difference in branching patterns, the bud scales of terminal buds are essentially the same in having a leaf base developed to function as a protecting organ. In all the sympodial species, during the abortion of a sympodium shoot tip, one or two pairs of primordia were found to occur on the apex, and later wither. These primordia resemble bud scales of terminal buds in their ontogeny and morphology, and appear to be rudimentary. It is suggested that a rudimentary terminal bud develops together with the establishment of sympodial branching, and that sympodial branching has originated from monopodial branching. Based on this proposed evolutionary trend, it is suggested thatAcer has moved from less shady habitats into shady habitats with monopodial branching (advantageous for vertical growth) changing into sympodial branching (advantageous for lateral spread).     

In vitro regeneration of <Emphasis Type="Italic">Carlina acaulis</Emphasis> subsp. <Emphasis Type="Italic">simplex</Emphasis> from seedling explants

The aim of the study was to obtain an efficient system for Carlina acaulis subsp. simplex propagation. The experimental materials were shoot tips, fragments of hipocotyls, cotyledons and roots isolated from 10-day-old seedlings. The explants were transferred to the proliferation medium supplemented with different types of cytokinin: BA (13.3 μM), kinetin (13.9 μM) and zeatin (13.7 μM) in combination with NAA (0.54 μM). The best morphogenetic response was observed when explants were cultured on the BA supplemented medium. The maximum shoot organogenesis frequency was observed for shoot tip (nearly 94%). On average 8.6 axillary shoots were induced per explant. Multiplication rate increased during the first three subcultures. The shoots revealed a wide range of morphogenetic responses. Differences were observed in the presence or absence of hair on the surface of lamina. These changes had epigenetic character and were the effect of changes in DNA methylation, which is shown by differences in methylation pattern between 18S rRNA and 25S rRNA genes in the analyzed regenerated plants. Nearly 94% of plantlets were rooted on auxin lacking medium. Addition of auxin (NAA or IAA) increased both the rooting percentage (100%) and the number of roots per shoot, but their growth was inhibited. Shortening of the auxin exposition time reduced the number of roots. Moreover, high efficiency (90%) was observed for ex vitro rooting. Plantlets with a large number of roots survived better than the ones with only a few roots. Plants were able to flower and gave viable seeds.     

THE CULTURE OF APICAL BUDS OF XANTHIUM AND THEIR USE AS A BIOASSAY FOR FLOWERING ACTIVITY OF ECDYSTERONE

Conditions were developed for the sterile culture of shoot tips of Xanthium pensylvanicum Wallr. for use as a bioassay for flower-controlling chemicals. By using a modified Murashige-Skoog medium (minus the auxin but including kinetin) and light intensity much higher than usual for plant tissue cultures, fast growth and development of the shoot tips was achieved. Under short-day conditions (8 hr day: 16 hr night), the cultures from vegetative shoots flowered and fruited; under noninductive conditions (using a 2 hr light-break in the middle of the dark period), the shoot tips continued vegetative development. Both intact plants and cultured tips could be photoinduced in the first days after germination. Ecdysterone, a potent insect moulting hormone, was tested in the bioassay system. It was without either qualitative or quantitative effect on flowering or vegetative development on either cultured shoot tips or intact plants irrespective of whether they were under inductive or noninductive photoperiodic conditions.     

Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

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.     

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.