Morphogenesis of potato plants in vitro. II. Endogenous levels,distribution, and metabolism of IAA and cytokinins

The levels of endogenous IAA and cytokinins (zeatin, zeatin riboside, isopentenyladenine, and isopentenyladenosine) were determined in potato plants cultured in vitro under red light (R) and blue light (B) on medium with or without hormones. On medium without hormones in B, plants contained much higher cytokinin levels, particularly in leaves and roots, and also slightly elevated IAA levels. Kinetin in the medium in B changed the distribution of cytokinins and significantly increased IAA level in roots. In R, the presence of kinetin led to an increased cytokinin level in the whole plant, while the IAA level was slightly lower. IAA in the medium in B decreased cytokinin level in all plant parts, while the IAA level did not change significantly. In R, the presence of IAA in the medium led to a moderate increase of CK level and to a significant increase in IAA level, especially in roots. Uptake of 1-¹⁴C-IAA and of ³H-zeatin was generally higher in B than in R. Higher percentage of IAA taken up in B was converted to conjugates in the roots. Metabolism of ³H-zeatin was similar in R and B with only slight differences in metabolite amounts.Thus, in all experimental situations in which tuber formation was stimulated, IAA level in roots and stolons rose significantly, stressing the importance of an IAA gradient for tuber formation.     

Agrobacterial <Emphasis Type="Italic">rol</Emphasis> genes modify thermodynamic and structural properties of starch in microtubers of transgenic potato

Wild-type (WT) plants of potato (Solanum tuberosum L.) and their transgenic forms carrying agrobacterial genes rolB or rolC under the control of B33 class I patatin promoter were cultured in vitro on MS medium with 2% sucrose in a controlled-climate chamber at 16-h illumination and 22°C. These plants were used as a source of single-node stem cuttings, which were cultured in darkness on the same medium supplemented with 8% sucrose. The tubers formed on them were used for determination of the structure of native starch using the methods of differential scanning microcalorimetry (DSC), X-ray scattering, and scanning electron microscopy. It was found that, in starch from the tubers of rolB-plants, the temperature of crystalline lamella melting was lower and their thickness was less than in WT potato. In tubers of rolC plants, starch differed from starch in WT plants by a higher melting temperature, considerably reduced melting enthalpy, and a greater thickness of crystalline lamellae. Deconvolution of DSC thermogram makes it possible to interpret the melting of starch from the tubers of rolC plants as the melting of two independent crystalline structures with melting temperatures of 65.0 and 69.8°C. Electron microscopic examination confirmed the earlier obtained data indicating that, in the tubers of rolC plants, starch granules are smaller and in the tubers of rolB plants larger than in WT plants. Possible ways of influence of rol transgenes on structural properties of starch in amyloplasts of potato tubers are discussed.     

Hormonal regulation of tuber formation in potato plants

Tuber formation is a complex process comprising several stages: stolon formation and growth, induction of tuberization, tuber initiation, and tuber growth. This review considers successive stages of tuber formation and their hormonal regulation. Special attention is paid to the effects on tuber formation of such phytohormones as gibberellins, cytokinins, jasmonic acid, and auxins. Physiological and some molecular-genetic aspects of their action on tuber photoperiodic induction and initiation are discussed.     

Effect of indole-3-acetic acid and kinetin on tuberisation parameters of different cultivars and transgenic lines of potato in vitro

The effect of indole-3-acetic acid or kinetin on the weight and numberof microtubers formed was studied on single node cuttings of sevendifferent potato (Solanum tuberosum L.) cultivars as well astransgenic lines harbouring rolB or rolC genes undercontrol of the patatin class I (B33) promoter. Plants were cultivatedin vitro in the dark on solidified MS medium containing 1 to8% sucrose with or without phytohormones. Most of thenontransformed potato cultivars and transgenic lines responded tohormone application by an increase in tuber yield. Auxin and cytokininacted differently: IAA increased predominantly the tuber size whilekinetin increased the number of tubers. RolC transformantsdisplayed an altered response to sucrose and especially to auxin. Thedegree of phytohormone effect on tuberisation parameters depended onsucrose content of the medium and potato genotype.     

Interaction between day length and phytohormones in the control of potato tuberization in the in vitro culture

We studied the interaction of the day length, cytokinins, and gibberellins in the control of tuberization in potato (Solanum tuberosum L, cv. Desire) plants and derived transgenic plants with the inserted PHYB gene from Arabidopsis encoding the synthesis of phytochrome B apoprotein and put under the control of the 35S CaMV promoter. Plantlets were cultured in vitro on hormone-free MS medium containing 5% sucrose and kinetin (1 mg/l) or/and GA (0.5 and 1.0 mg/l), at long day (LD, a 16-h photoperiod), short day (SD, a 10-h photoperiod), or continuous darkness conditions. The content of cytokinins (Ck, zeatin, and zeatin riboside) in various plant organs was determined by the immunoenzyme method, and GA activity was measured in bioassay with dwarf pea. Potato plant transformation with the PHYB gene enhanced substantially tuber initiation inhibition by LD. Kinetin addition to culture medium enhanced tuberization and reduced Ck content in aboveground shoots and Ck redistribution in the favor of underground organs. GA addition to the culture medium suppressed tuberization and induced Ck accumulation in aboveground organs. We concluded that Ck role in tuberization depends on their predominant localization in above- or underground potato organs. The involvement of Ck and GA in the competitive relations between growing tubers and shoots is considered.     

Photoperiodic and Hormonal Control of Tuberization in Potato Plants Transformed with the <Emphasis Type="Italic">PHYB</Emphasis> Gene from <Emphasis Type="Italic">Arabidopsis</Emphasis>

We studied photoperiodic and hormonal regulation of tuberization in wild-type potato (Solanum tuberosum L., cv, Desiree) plants and derivative transgenic plants harboring the PHYB gene from Arabidopsis, which encodes the phytochrome B apoprotein, under the control of the cauliflower mosaic virus 35S promoter. Plants were cultured on hormone-free Murashige and Skoog nutrient medium containing 5% sucrose or on the same medium supplemented with 1 mg/l kinetin under conditions of long day (LD, 16 h), short day (SD, 10 h), or SD with interrupted long night. We estimated cytokinins (zeatin and zeatin riboside) in underground and aboveground plant organs by the ELISA technique and GA activity in a bioassay with dwarf pea seedlings. Under LD conditions, transgenic plants produced substantially less tubers than wild-type plants. Kinetin addition to the culturing medium resulted in stimulation of tuberization under LD conditions, especially pronounced in the PHYB plants. The content of cytokinins and the activity of GA were much higher under LD conditions, especially in leaves. The total level of both phytohormones was higher in transformed as compared to wild-type plants. A relation of phytochrome-dependent tuberization to the hormonal status of underground and above-ground plant organs and possible reasons for kinetin stimulatory effect on this process are discussed.     

Growth pattern, tuber formation and hormonal balance in in vitro potato plants carrying ipt gene

Nodal cuttings of in vitro grown potato plants (Solanum tuberosum, cv. Miranda) were transformed by a vector plasmid carrying ipt gene of Agrobacterium tumefaciens. From the initial teratoma stage 5 clones of transgenic plants (1, 2, 11, 13 and 15) were obtained, which displayed in varying degree shortening of the internodes, decrease of the leaf size, decrease of apical dominance and poor rooting. In addition, two of the clones (11 and 13) showed increased stolon and tuber formation. In all these clones the endogenous level of free cytokinins (CKs) was increased: from 40% in clone 11 to almost 300% in clone 1. Also free indole-3-acetic acid (IAA) level was increased, but to a lower degree; the maximal increase was 160% (clone 13). Applied kinetin or IAA (1 mg·l^-1) strongly suppressed root and tuber formation in clones 11 and 13, although they did not affect or even stimulated these processes in control plants. For control plants the minimal medium sucrose concentration necessary for tuber initiation was 6% whereas in clone 11 plants 2% was sufficient. Different distribution of endogenous CKs and IAA was observed in clone 11 and control plants. The highest CK content was found in transgenic plants in stems and in controls in leaves. In clone 11 plants abscisic acid (ABA) level was significantly increased in comparison to the control throughout the cultivation period. Ethylene formation was strongly increased the first week after the subcultivation and later on the difference between transgenic and control plants rapidly diminished. Reactions of clone 11 plants to red (RL) and blue light (BL) were similar to reactions of control plants. In RL clone 11 plants were tall and thin with stunted leaves; in BL they had a teratoma-like appearance and formed a very high number of tubers. The role of hormones in these changes in growth and tuber formation is discussed.     

Tuber Formation and Growth of in vitro Cultivated Transgenic Potato Plants Overproducing Phytochrome B

We studied the effect of the ectopic expression of the Arabidopsis PHYB gene, which encodes the phytochrome B (phyB) apoprotein, under the control of cauliflower mosaic virus 35S promoter on the photoperiodic response of tuberization and growth of potato (Solanum tuberosum L., cv. Désirée) transformed lines. Stem cuttings of transformed and control plants were cultured on Murashige and Skoog nutrient medium containing 5 or 8% sucrose in the phytotron chambers at 20°C under conditions of a long day (16 h), a short day (10 h), or in darkness. We showed that the overexpression of the PHYB gene enhanced the inhibitory effect of the long day on tuberization. In addition, tuber initiation in these transformed plants occurred at a higher sucrose concentration. The insertion of the PHYB gene decreased plant and tuber weights and shortened stems and internodes. Thus, we demonstrated the complex result of the PHYB gene insertion: it affected the photoperiodic response of tuberization, the control of tuber initiation by sucrose, and the growth of potato vegetative organs.