S. A. Golyanovskaya

Morphogenesis of potato plants in vitro. I. Effect of light quality and hormones

Stem cuttings of potato plants (Solanum tuberosum L., cv. Miranda) were cultured in vitro on MS medium with sucrose either without or with addition of indole-3-acetic acid (IAA) or kinetin (K) under red light (R) or blue light (B). Plants on medium without hormones under R were thin, long, with very small leaves, and produced no or only a few microtubers (after longer-lasting cultivations). In B, plants remained short, thick, with large, wellde-veloped leaves and produced a significant amount of microtubers. Darkening of both roots and shoots strongly promoted tuber formation; the tubers were formed on the darkened part of the plant. IAA had no pronounced effect on plant development in B except for slight lengthening of the stem, and, in longer cultivations, slightly enhanced tuber formation as well. In R, IAA brought about several significant effects: stem reduction and induction of tuber formation being the most significant. Kinetin in R increased tuber formation slightly. In B, kinetin not only strongly stimulated tuber formation, but also increased the total fresh weight and root (+ stolons)/shoot ratio. Results are discussed with regard to the possible role of auxins and/or cytokinins in mediating the morphogenetic effects of light.

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.

Regulation of potato tuber dormancy and sprouting

Dormancy is the final stage of tuber life serving to preserve tubers as organs of vegetative reproduction under unfavorable growth conditions. Since the duration of potato tuber dormancy and their sprouting time have significant economic importance, much attention is given to the study of the regulation of these processes. This review considers metabolite, genetic, and hormonal aspects of regulation of potato (Solanum tuberosum L.) tuber dormancy and sprouting. Particular attention is paid to the relationship between processes occurring in different parts of the tuber: its storage tissues and buds. The interaction of hormonal and metabolite (carbohydrate) regulation of dormancy and sprouting is discussed.

Effect of Rol Transgenes, IAA, and Kinetin on Starch Content and the Size of Starch Granules in Tubers of In Vitro Potato Plants

Stem cuttings were produced from Solanum tuberosum L., cv. Desiree, plants and their transgenic forms harboring rolB and rolC genes from Agrobacterium rhizogenes. Plants were cultured on hormone-free Murashige and Skoog nutrient medium (MS) and on MS supplemented with IAA or kinetin. In microtubers developed on these cuttings, we estimated the content of starch and the number and size of starch granules. Expression of rol genes changed these indices: in tubers of rolC transformants, a greater number of small granules were produced, whereas in tubers of rolB transformants, a fewer number of large granules were developed as compared with wild-type plants. Expression of rol genes did not affect starch content during the first three weeks of cutting culturing but increased it by 15–30% in five-week-old tubers. IAA addition to MS medium increased starch content and the size of starch granules in control plants and rolB tubers by 10–30%, whereas kinetin did not exert any significant influence. The effects of rol transgenes on the initiation and termination of starch granule development are discussed.

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.

Introduction of the Arabidopsis PHYB gene increases resistance of photosynthetic apparatus in transgenic Solanum tuberosum plants to UV-B radiation

The influence of phytochrome B overproduction in 50- to 60-day-old transgenic potato plants (Solanum tuberosum L., lines Dara 5 and Dara 12 with moderate and intense expression of PHYB, respectively) on the resistance of photosynthetic apparatus to UV-B irradiation was investigated. In plants unexposed to UV-B, there was no significant difference in photosynthetic rates (Pn) and fluorescence parameters (Fv/Fm, qN, qP) between the nontransformed (NT) line and Dara-5 and Dara-12 lines, whereas the content of photosynthetic pigments per 1 cm2 leaf area was higher in the transgenic plants. Irradiation with UV-B resulted in the decrease in photosynthetic rate in NT plants by 35–45%, whereas in Dara-12 line this rate was lowered by only 20–25%. Exposure to UV-B reduced the amplitudes of both fast and slow components of delayed fluorescence (DF), which indicated the diminished efficiency of photosystem II (PSII). The decrease in the maximum amplitude of the slow DF component was markedly lower in Dara-12 (19%) than in NT line (33%). The maximal photochemical quantum yield of PSII (Fv/Fm ratio) in plants exposed to UV-B was also suppressed stronger in NT line than in Dara-12. The line Dara-5 had intermediate position among other lines in terms of UV-B resistance of photosynthesis and PSII activity, but it was closer to NT than to Dara-12. Thus, the potato plants actively expressing the gene of Arabidopsis phytochrome B apoprotein (PHYB) demonstrated a higher resistance of photosynthetic apparatus to UV-B radiation compared to nontransformed plants. The elevated UV-B tolerance is most likely related to the increased leaf content of chlorophyll, carotenoids, and flavonoids.

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.

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.

Photoperiodic and Hormonal Control of Tuberization in Potato Plants Transformed with the PHYB Gene from Arabidopsis

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.

An improved tolerance of PHYB-transgenic potato plants to the middle-wave ultraviolet irradiation.

Phytochrome is one of the basic plant photoreceptors involved in the photoregulation of many morphological and biochemical processes [1]. The practical importance of many phytochrome-controlled morphogenetic processes stimulated finding and active investigation of several phytochrome proteins. Potato morphogenesis is one of such processes [2]. The constitutive expression of the gene for phytochrome B apoprotein ( PHYB ) from Arabidopsis in potato plants has a pleiotropic effect and results in the development of short, very branched plants with active photosynthesis and increased levels of chlorophyll and anthocyanin [3, 4]. As a result, these transgenic plants with increased phytochrome B content were more tolerant to the long-term high light irradiation. Under these conditions, in transgenic potato plants, photosynthesis was less suppressed and chlorophyll degradation was retarded as compared to wild-type plants [3]. It was reported that the phototolerance of the photosynthetic apparatus [5] and the accumulation of phenolic compounds [6], anthocyanins in particular, improved plant tolerance to UV radiation. This allowed us to suggest that potato plants with increased phytochrome B content might be more tolerant not only to high light but also to UV radiation [3].