The role of polyamines during <Emphasis Type="Italic">in vivo</Emphasis> and <Emphasis Type="Italic">in vitro</Emphasis> development

Polyamines are ubiquitous polycationic compounds that mediate fundamental aspects of cell growth, differentiation, and cell death in eukaryotic and prokaryotic organisms. In plants, polyamines are implicated in a variety of growth and developmental processes, in addition to abiotic and biotic stress responses. In the last decade, mutant studies conducted predominantly in Arabidopsis thaliana revealed an obligatory requirement for polyamines in zygotic and somatic embryogenesis. Moreover, our appreciation for the intricate spatial and temporal regulation of intracellular polyamine levels has advanced considerably. The exact molecular mechanism(s) through which polyamines exert their physiological response remains somewhat enigmatic and likely serves as a major area for future research efforts. In the following review, we discuss recent advances in the plant polyamine field, which range from metabolism and mutant characterization to molecular genetics and potential mode(s) of polyamine action during growth and development in vitro and in vivo. This review will also focus on the specific role of polyamines during embryogenesis and organogenesis.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Effect of polyamines on in vitro somatic embryogenesis in <Emphasis Type="Italic">Momordica</Emphasis><Emphasis Type="Italic">charantia</Emphasis> L.

The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli (5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous free PA levels.     

Expression analysis and genetic mapping of three <Emphasis Type="Italic">SEPALLATA</Emphasis>-like genes from peach (<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch)

SEPALLATA (SEP) MADS box genes play essential and diverse roles in reproductive organ development. To investigate the SEP gene function in peach we isolated three SEP-like genes, PrpMADS2, PrpMADS5, and PrpMADS7, which belong to distinct SEP gene clades. They appeared as single copy genes in the peach genome and were found to preferentially express in flowers and fruits. Arabidopsis transformants expressing 35S: PrpMADS2 were indistinguishable from wild-type plants. Overexpression of PrpMADS5 led to earlier flowering. Through chimeric repressor silencing technology, PrpMADS5 was found to function in floral organ development. Expression of PrpMADS7 in Arabidopsis caused a dramatic attenuation of both juvenile and adult growth phases and, in severely affected plants, it led to flower formation immediately after the embryonic phase. Two microsatellite markers were developed for PrpMADS2 and PrpMADS5 and assigned to the genetic linkage groups 5 and 1, respectively. PrpMADS7, previously identified as PrpAGL2, and PrpMADS5 were identified as potential loci to modify the flowering time and floral organs in Prunus species. Moreover, our results showed the diversification of SEP genes in peach. The gene sequences have been deposited in GenBank and will appear under the accession numbers BQ102369, EF440351, and EF440352.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

Characterization of <Emphasis Type="Italic">HoMADS 1</Emphasis> and its induction by plant hormones during <Emphasis Type="Italic">in vitro</Emphasis> ovule development in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

To understand the molecular mechanism of ovule development, a MADS box gene,HoMADS 1, has been isolated from the ovule tissues of Hyacinthus. Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.     

Comparison of gene expression under <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">ex vitro</Emphasis> conditions during rooting of grape cuttings through mRNA differential display

An mRNA differential display (DD) analysis during rooting in grape cuttings was carried out to determine whether gene expression patterns differed under in vitro and ex vitro conditions. The four tissue samples for differential display and subsequent Northern hybridization analyses included control stem tissue from in vitro and ex vitro sources, microcuttings planted in MS based in vitro rooting medium and softwood cuttings planted in ex vitro soil medium, both collected 48 h after planting. DD autoradiographs showed gross similarity in banding pattern between in vitro and ex vitro stem tissue, whether constitutive or induced. Northern blot analysis of a few bands that appeared to be differentials did not indicate them as true positives. The results suggested that gene expression pattern during physiological processes such as rooting may be identical under in vitro and ex vitro conditions.     

Highly efficient <Emphasis Type="Italic">in vitro</Emphasis> adventitious shoot regeneration of peppermint (<Emphasis Type="Italic">Mentha</Emphasis> x <Emphasis Type="Italic">piperita</Emphasis> L.) using internodal explants

An in vitro regeneration system with a 100% efficiency rate was developed in peppermint [Mentha x piperita] using 5- to 7-mm-long second internode stem segments of 3-wk-old stock plants. Shoots developed at sites of excision on stem fragments either directly from the cells or via primary calluses. The optimal medium for maximum shoot initiation and regeneration contained Murashige and Skoog (MS) salts, B5 vitamins, thidiazuron (TDZ, 11.35 μM), ZT (4.54 μM), 10% coconut water (CW), 20 g l⁻¹ sucrose, 0.75% agar, adjusted to pH 5.8. A frequency of 100% shoot initiation was achieved, with an average of 39 shoots per explant. This regeneration system is highly reproducible. The regenerated plants developed normally and were phenotypically similar to Black Mitcham parents.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

Propagation of <Emphasis Type="Italic">Haemaria discolor</Emphasis> via <Emphasis Type="Italic">in vitro</Emphasis> seed germination

In vitro propagation protocol for Haemaria discolor (Ker) Lindl. var. dawsoniana by artificial cross-pollination and asymbiotic germination of seeds has been developed. Fruit set (100 %) was obtained when the pollinia and ovules of various aged flowers were used for pollination. In vitro germination of seeds obtained from capsules of various ages was achieved on half-strength Murashige and Skoog’s (MS) medium supplemented with 3 % sucrose and 0.85 % agar. The germinated seedlings were cultured on half-strength MS medium with 0.2 % activated charcoal, 8 % banana homogenate, 0.1 mg dm⁻³ 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea (TDZ) and 1 mg dm⁻³ α-naphthaleneacetic acid (NAA). Ninety-six percent of plantlets survived after hardening in greenhouse.This research was supported by grant (91AS-3.1.1-CI-C3) from the Council of Agriculture, Executive Yuan of Taiwan and grants (NSC-89-2317-B055-002 and NSC-91-2317-B324-001) from the National Science Council of Taiwan. This paper is Agricultural Research Institute Contribution No. 2158.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Pelecyphora aselliformis</Emphasis> erhenberg (cactaceae)

Summary In vitro propagation of Pelecyphora aselliformis, a Mexican cactus which is considered rare and is highly valued in the commercial market, was initiated using seeds as explants. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium containing 8.8 μM benzyladenine (BA) or 4.6 μM kinetin at pH 7.0. After 120 d, each explant gave rise to five shoots and this number of shoots increased 20–25% after subculture. The hyperhydricity was similar in both media, but callus formation was lower on the medium with BA. The shoot development, in terms of epicotyl length, and fresh and dry weight after 6 wk, was also recorded. The epicotyl length was similar on shoot-forming media but the quality of shoots was better on media containing BA. In about 1 yr, 500–600 well-defined shoots were obtained. The rooting of shoots was very slow and a vigorous radical system was observed after 1 yr of culture.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

Exogenous sucrose can decrease <Emphasis Type="Italic">in vitro</Emphasis> photosynthesis but improve field survival and growth of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis> plantlets

Summary Coconut (Cocos nucifera L.) plantlets grown in vitro often grow slowly when transferred to the field possibly, due to a limited photosynthetic capacity of in vitro-cultured plantlets, apparently caused by the sucrose added to growth medium causing negative feedback for photosynthesis. In this paper, we tested the hypothesis that high exogenous sucrose will decrease ribulose 1,5-bisphosphate carboxylase (Rubisco) activity and photosynthesis resulting in limited ex vitro growth. Plantlets grown with high exogenous sucrose (90 gl⁻¹) had reduced photosynthetic activity that resulted in a poor photosynthetic response to high levels of light and CO₂. These plantlets also had low amounts of Rubisco protein, low Rubisco activity, and reduced growth despite showing high survival when transferred to the field. Decreasing the medium’s sucrose concentration from 90 to 22.5 gl⁻¹ or 0 gl⁻¹ resulted in increased photosynthetic response to light and CO₂ along with increased Rubisco and phosphoenolpyruvate carboxylase (PEPC) activities and proteins. However, plantlets grown in vitro without exogenous sucrose died when transferred ex vitro, whereas those grown with intermediate exogenous sucrose showed intermediate photosynthetic response, high survival, fast growth, and ex vitro photosynthesis. Thus, exogenous sucrose at moderate concentration decreased photosynthesis but increased survival, suggesting that both in vitro photosynthesis and exogenous sucrose reserves contribute to field establisment and growth of coconut plantlets cultured in vitro.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Kniphofia leucocephala</Emphasis>: influence of cytokinins

The role of cytokinins in the promotion of flowering in the endangered species Kniphofia leucocephala Baijnath. was investigated using shoots maintained in culture for 3 years. The highest percentage flowering (65%) was obtained on media containing 20 μM benzyladenine (BA). The inclusion of isopentenyladenine and zeatin in the media also resulted in flowering, but these treatments were less effective than BA in inducing flowering. The effect of cytokinins on flowering was dose-dependent, with high concentrations of BA inhibiting flower formation. Treatments that resulted in rooting of explants produced no flowers. The resulting inflorescences in all treatments did not mature and senesced prematurely, even when gibberellic acid (GA₃) was applied post-flower-emergence.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

Transgenic peach plants (<Emphasis Type="Italic">Prunus persica</Emphasis> L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an <Emphasis Type="Italic">in vivo</Emphasis> marker

The main obstacle to genetic engineering of fruit tree species is the regeneration of transformed plantlets. Transformation events in peach (Prunus persica L.) have been reported using particle bombardment or Agrobacteriummediated transformation of immature embryos. However, the regeneration of plants from transgenic tissues is still difficult and the recovery of non-chimeric plants has not been reported to date. In this paper we describe an efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. This represents an important advantage due to the availability of such material throughout the year. A. tumefaciens strain C58 (pMP90) containing the binary plasmid pBin19 was used as vector system for transformation. We used the Nospro-nptII-Noster cassette as a selectable marker and the CaMV35Spro-sgfp-CaMV35Ster cassette as a vital reporter gene coding for an improved version of the green fluorescent protein (sGFP). In vitro cultured embryo sections were Agrobacterium-cocultivated and, after selection, transgenic shoots were regenerated. Shoots that survived exhibited high-level of sGFP expression mainly visible in the young leaves of the apex. In vivo monitoring of GFP expression permitted an early, rapid and easy discrimination of both transgenic and escape buds. After elimination of escapes, transgenic shoots were rooted in vitro and the recovered plantlets were screened using PCR amplification. Southern analysis confirmed stable genomic integration of the sgfp transgene. The high levels of GFP expression were also maintained in the second generation of transgenic peach plants.     

Thidiazuron Promotes <Emphasis Type="Italic">In Vitro</Emphasis> Plant Regeneration of <Emphasis Type="Italic">Arachis correntina</Emphasis> (<Emphasis Type="Italic">Leguminosae</Emphasis>) via Organogenesis

Arachis correntina (Burkart) Krapov. & W.C. Gregory is a herbaceous perennial leguminous plant growing in the Northeast of the Province Corrientes, Argentina. It is important as forage. The development of new A. correntina cultivars with improved traits could be facilitated through the application of biotechnological strategies. The purpose of this study was to investigate the plant regeneration potential of mature leaves of A. correntina in tissue culture. Buds were induced from both petiole and laminae on 0.7% agar-solidified medium containing 3% sucrose, salts and vitamins from Murashige and Skoog (MS) supplemented with 0.5–25 M thidiazuron (TDZ). Shoot induction was achieved by transference of calli with buds to MS supplemented with 5 M TDZ. Fifty-four percent of the regenerated shoot rooted on MS + 5 M naphthaleneacetic acid. Histological studies revealed that shoots regenerated via organogenesis.     

Induction of streptomycin-resistant plantlets in <Emphasis Type="Italic">Solanum surattense</Emphasis> through <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis

The species Solanum surattense Burm.f. has importance in ayurvedic medicine and also as vegetable. Streptomycin-resistant plantlets were induced showing chloroplast encoded mutants in S. surattense from mutagenised (ethyl methane sulphonate and gamma-rays) cotyledon explants. Chloroplast encoded – streptomycin resistant – shoots were developed from green (unbleached) sectors of the cotyledons. The streptomycin-resistant plants were similar to parental plants in morphology and ploidy level (2n=2x=24). Reciprocal crosses between streptomycin-resistant and the original streptomycin sensitive plants have shown the non-Mendelian transmission under the control of chloroplast – DNA. These antibiotic resistant plants are useful in designing biochemical selection schemes aimed at somatic hybrid/cybrid recovery in S. surattense.