In vitro assay of native Iranian almond species (<Emphasis Type="Italic">Prunus</Emphasis> L. spp.) for drought tolerance

Eight native Iranian almond species from three sections, ‘Euamygdalus’ (Prunus communis; Prunus eleagnifolia and Prunus orientalis); ‘Lycioides’ (Prunus lycioides and Prunus reuteri) and ‘Spartioides’ (Prunus arabica, Prunus glauca and Prunus scoparia) were in vitro screened for drought tolerance using sorbitol and polyethylene glycol (PEG) as an osmoticum. Different levels of water stress were induced using five concentrations of either sorbitol or polyethylene glycol in Woody Plant Medium (WPM). Water potential of various media ranged from −0.80 to −2.05 MPa and water stress in culture medium adversely affected plantlet growth. Wild species from ‘Spartioides’ were less affected than ‘Lycioides’ and ‘Euamygdalus’. At the same level of water potential, sorbitol had lower adverse effects than PEG; the latter being severe. Prunus × sorbitol and Prunus × PEG interactions were significant. At 0.2 M sorbitol and 0.003 M PEG, ‘Spartioides’ produced significantly more roots with higher total root length and root volume, as well as root-dry weight than those of ‘Lycioides’ and ‘Euamygdalus.’ It is concluded that in vitro screening of native Iranian almond species under specific and limited water-stress conditions may provide a system for effectively differentiating the wild species of almond for their expected root mass production under field conditions.     

Regeneration of <Emphasis Type="Italic">citrus sinensis</Emphasis> (+) <Emphasis Type="Italic">clausena lansium</Emphasis> intergeneric triploid and tetraploid somatic hybrids and their identification by molecular markers

Summary Somatic hybrid plants were regenerated via electrofusion between leaf-derived protoplasts of ‘Chicken heart’ sweet wampee (Clausena lansium) and embryogenic protoplasts of ‘Newhall’ navel orange (Citrus sinensis Osbeck). Most of the complete plantlets were formed via mini-grafting. Flow cytometry showed that most of the regenerants were tetraploids as expected, but unexpectedly three plantlets were triploids. Simple sequence repeat (SSR) analysis of seven randomly selected tetraploids and the three triploids showed that they had specific fragments from both fusion parents, thereby confirming their hybridity. Analysis of cytoplasmic genomes using universal primers revealed that their chloroplast DNA (cpDNA) band pattern was identical to the mesophyll parent, while their mitochondrial genomes were of the navel orange type. According to the SSR results, the triploids obtained in this study were most likely due to chromosome elimination of ‘Chicken heart’ sweet wampee prior to plant regeneration.     

The establishment of cell suspension cultures ofGladiolus that regenerate plants

Summary Inflorencence stalks from greenhouse-grownGladiolus plants of the cultivars ‘Blue Isle’ and ‘Hunting Song’ cultured on a Murashige and Skoog basal salts medium supplemented with 53.6 μM 1-napthaleneacetic acid formed a compact, not friable type of callus that regenerated plantlets. Cormel slices and intact plantlets of three cultivars (‘Peter Pears’, ‘Rosa Supreme’, ‘Jenny Lee’) propagated through tissue culture formed a friable type of callus when cultured on Murashige and Skoog basal salts medium supplemented with 2,4-dichlorophenoxyacetic acid. This friable callus readily formed a cell suspension when the callus was placed in a liquid medium. Plants were regenerated from two-month-old suspension cell cultures of the commercial cultivar ‘Peter Pears’ after the suspension cells had been cultured on solid medium.     

High-resolution genetic map of the <Emphasis Type="Italic">Rvi15</Emphasis> (<Emphasis Type="Italic">Vr2</Emphasis>) apple scab resistance locus

The Rvi15 (Vr2) apple scab resistance locus found in the GMAL 2473 accession has been previously mapped to the top of the Linkage Group 2 (LG2) by analyzing 89 progeny plants of a cross between ‘Idared’ and GMAL 2473. A new population of 989 progeny plants, derived from a cross between ‘Golden Delicious’ and GMAL 2473, has been analyzed with the two SSR markers CH02c02a and CH02f06, previously found to be associated with Rvi15 (Vr2), and with two published markers derived from NBS sequences (ARGH17 and ARGH37) estimated to map close to the Rvi15 (Vr2) locus. ARGH17 and ARGH37, were found to be the closest markers to the resistance locus, bracketing it within an interval of 1.5 cM. The SSRs mapped one on each side of Rvi15 (Vr2). CH02f06 mapped at 2.9 cM from ARGH37 while CH02a02a mapped at 1.7 from ARGH17. The position of Rvi15 (Vr2) respect to CH02a02a indicates that Rvi15 (Vr2) and Rvi4 (Vh4), a second apple scab gene mapped on the top of LG2, are two different resistance genes. In order to develop even more tightly linked markers to Rvi15 (Vr2), ARGH17 was used as the starting point for chromosome walking through the Rvi15 (Vr2) homolog region of the cv. ‘Florina’. A single ‘Florina’ BAC clone, 36I17, was sufficient to span the homologous locus in the new population’s recombinant progeny. Sequencing of the 36I17 BAC clone allowed identifying seven putative ORFs, including two showing a TIR-NBS-LRR structure. Ten additional markers could be developed mapping within a 1.8 cM interval around the Rvi15 (Vr2) resistance gene. ARGH17 and GmTNL1 markers, the latter also derived from NBS-LRR resistance gene homolog sequence, are the closest markers to Rvi15 (Vr2) bracketing it within a 0.5 cM interval. The availability of 12 markers within the Rvi15 (Vr2) region, all within a small physical distance (kbp) in ‘Florina’, suggests that cloning of the Rvi15 (Vr2) apple scab resistance gene from GMAL 2473 will be possible.     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.     

Flower Bud Culture of Shallot (Allium cepa L. Aggregatum group) with Cytogenetic Analysis of Resulting Gynogenic Plants and Somaclones

Unpollinated flower culture was applied for induction of gynogenesis and somatic organogenesis in three shallot strains, ‘Dili-white’, ‘Yogya’ and ‘Dili-red’, from Indonesia. Chromosome surveys were performed on the plants obtained. From a total of 6,812 flowers, 89 plantlets were obtained by gynogenesis, of which 10 could be acclimated. Most of the plantlets were induced from ‘Dili-white’. Of the gynogenetic plants examined, two were haploid (2n = 8), four were naturally doubled haploid (2n = 16) and the remaining four were mixoploid (2n = 8, 16 or 2n = 16, 32, 64). ‘Dili-red’ showed the highest frequency of somatic organogenesis. Fifty-nine directly regenerated plants and 293 callus-derived plants were obtained from somatic organogenesis from the cultured flowers. Based on the chromosome number, frequencies of somaclonal variation were high both in the directly regenerated plants and the callus-derived plants. The frequency of tetraploid plants (2n = 32) in the former (50%) was higher than in the latter (33%). From these results we conclude that unpollinated flower culture is an effective method for chromosome doubling simultaneously with haploid induction in shallot.     

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (<Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.     

An efficient protocol for large-scale plantlet production from male floral meristems of <Emphasis Type="Italic">Musa</Emphasis> spp. cultivars Virupakshi and Sirumalai

In vitro propagation has played a key role for obtaining large numbers of virus free, homogenous plants, and for breeding of plantains and bananas (Musa spp.). Explant sources utilized for banana micropropagation include suckers, shoot tips, and floral buds. The present study employed male floral meristems as explant material for micropropagation of hill banana ecotypes (AAB) ‘Virupakshi’ and ‘Sirumalai.’ Immature male floral buds were collected from healthy plants from hill banana growing areas. Exposure of explants to ethyl alcohol (70%, v/v) for 30 s, then mercuric chloride (0.1%, w/v) for 30 s, followed by three independent rinses of 5 min each in autoclaved, double-distilled water satisfactorily reduced the contamination. Male floral bud explants were cultured on Murashige and Skoog (MS) basal medium supplemented with different combinations of 6-benzylaminopurine (BAP), coconut water, naphthaleneacetic acid, gibberellic acid, and additional supplements. MS medium supplemented with 5 mg l⁻¹ BAP and coconut water (15%) was the most efficient media for shoot initiation and multiple shoot formation (15 shoots from a single part of a floral bud). The best response for shoot elongation was obtained using the combination of basal MS, 5 mg l⁻¹ BAP, 1 mg l⁻¹ naphthaleneacetic acid and 1.5 mg l⁻¹ gibberellic acid. Regenerated shoots were rooted in basal MS medium within 15–20 d. The rooted plantlets were transferred to a soil mixture and maintained at a temperature of 25 ± 2°C for 10 d and then at room temperature (30–32°C) for 2 wk, before transferring to a greenhouse. The regenerated plantlets showed 100% survival.     

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented <Emphasis Type="Italic">Pelargonium capitatum</Emphasis> cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively.     

In vitro germplasm conservation of high Δ<Superscript>9</Superscript>-tetrahydrocannabinol yielding elite clones of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L. under slow growth conditions

Germplasm conservation of a high Δ⁹-tetrahydrocannabinol yielding variety of Cannabis sativa L. was attempted using synthetic seed technology and media supplemented with osmotic agents. Explants of nodal segments containing single axillary bud were excised from in vitro proliferated shoot cultures and encapsulated in high-density sodium alginate (230 mM) hardened by 50 mM CaCl₂. The ‘encapsulated’ (synthetic seeds) and ‘non-encapsulated’ nodal segments were stored at 5, 15 and 25°C for 8, 16 and 24 weeks and monitored for the re-growth and survival frequency under the tissue culture conditions (16-h photoperiod, 25°C) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ 0.5 μM). ‘Encapsulated’ nodal segments could be stored at low temperature 15°C up to 24 weeks with maximum re-growth ability and survival frequency of 60%. Similar to ‘encapsulated’ cultures, the highest re-growth in ‘non-encapsulated’ cultures was observed in the explants kept at 15°C without osmotic agents. Furthermore, the effect of osmotic agents mannitol and sorbitol (2 and 4% w/v, added individually and in combination to the media at culture room conditions i.e. 25°C) on non-encapsulated shoot cultures was also evaluated. A considerable decrease in re-growth and survival was observed in the cultures treated with osmotic agents. Among the cultures treated with different concentrations of osmotic agents, the highest rate of re-growth and survival was observed at the lowest concentration of 2% sorbitol and 2% mannitol individually added to the media. Well-developed plantlets regenerated from ‘encapsulated’ nodal segments were successfully acclimatized inside the growing room with 90% survival frequency. Gas chromatography-flame ionization detection (GC-FID) was used to compare the chemical profile and the concentration of the different cannabinoids (cannabidiol, cannabichromene, cannabigerol, cannabinol, Δ⁹-tetrahydrocannabinol and tetrahydrocannabivarin) of the plants grown from ‘encapsulated’ nodal segments to that of the donor plant. The data showed similar cannabinoid profile and insignificant differences in the cannabinoids content between the two types of plants. This study is of high significance since the encapsulation technology would allow the prolonged storage (thus reducing the cost of labor) of high-yielding C. sativa germplasm selected for the isolation of THC, a high-value bulk active pharmaceutic.     

Shoot organogenesis in leaf explants of <Emphasis Type="Italic">Hydrangea macrophylla</Emphasis> ‘Hyd1’ and assessing genetic stability of regenerants using ISSR markers

For the first time, an in vitro regeneration protocol of Hydrangea macrophylla ‘Hyd1’ was developed. Effects of different plant growth regulators (PGRs) on shoot regeneration were investigated jointly with selecting optimal basal media and cefotaxime concentrations. The highest frequency of shoot organogenesis (100%) and mean number of shoots per explant (2.7) were found on Gamborg B5 basal medium supplemented with 2.25 mg/l 6-benzyladenine (BA), 0.1 mg/l Indole-3-butyric acid (IBA), 100 mg/l cefotaxime and 30 g/l sucrose solidified by 7 g/l agar. Regenerated shoots were rooted by culturing on perlite plus half strength liquid B5 basal medium with 0.5 mg/l NAA. Rooted plantlets were transplanted to the greenhouse with 100% survival rate. Genetic stability of 32 plantlets (one mother plant and 31 regenerants) was assessed by 44 ISSR markers. Out of 44 ISSR markers, ten markers produced clear, reproducible bands with a mean of 5.9 bands per marker. The in vitro regeneration protocol is potentially useful for the genetic transformation of Hydrangea macrophylla ‘Hyd1’.     

Micropropagation of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. ‘Malagouzia’ and ‘Xinomavro’

The effects of six basal media on in vitro shoot proliferation of the greek grapevines Vitis vinifera L. cv. ‘Malagouzia’ and ‘Xinomavro’ were investigated. Galzy and Zlenco proved to be the most effective for ‘Malagouzia’ and ‘Xinomavro’, respectively. If only BA was present in the medium, shoot development was poor and the plantlets were chlorotic. When the medium was supplemented with BA and NAA, growth was enhanced. The best ratio (in μM) of growth regulators was 0.5/0.3 for ‘Malagouzia’, and 0.1/0.03 for ‘Xinomavro’, which resulted in the highest number of microshoots per explant and greatest proliferation rate. The development of ‘Malagouzia’ and ‘Xinomavro’ explants at 21±2 and 26±2°C was also investigated, revealing the higher temperature to be more effective. Regarding rooting, 0.5 μM IBA improved root formation at 26°C for ‘Malagouzia’ and 0.5 μM IBA at 21°C for ‘Xinomavro’. Moreover, 0.5 μM IBA resulted in a higher rooting percentage (>95%) and proved to be more beneficial for the overall morphological appearance of the plantlets of ‘Malagouzia’. After acclimatization, survival of microshoots cultivated in media with IBA was higher than those in NAA.     

In vitro and in vivo anti-allergic effects of ‘benifuuki’ green tea containing O-methylated catechin and ginger extract enhancement

‘Benifuuki’, a tea (Camellia Sinensis L.) cultivar in Japan, is rich in anti-allergic epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3″Me). ‘Benifuuki’ green tea and simultaneous addition of ginger extract remarkably suppressed cytokine (TNF-α and MIP-1α) secretion from mouse bone marrow-derived mast cells after antigen stimulation and, as expected, suppressed delay-type allergy. After drinking ‘benifuuki’ green tea containing 43.5 mg of EGCG and 8.5 mg of EGCG3″Me, the AUC (area under the drug concentration time curve; min μg/ml) of EGCG was 6.72 ± 2.87 and EGCG3″Me was 8.48 ± 2.54 in healthy human volunteers. Though the dose of EGCG was 5.1 times the dose of EGCG3″Me, the AUC of EGCG3″Me was higher than that of EGCG. A double blind clinical study on subjects with Japanese cedar pollinosis was carried out. At the 11th week after starting the study, in the most severe cedar pollen scattering period, symptoms, i.e., blowing the nose and itching eyes, were significantly relieved in the ‘benifuuki’ intake group compared with the placebo group, and blowing the nose, itching eyes and nasal symptom score, and at the 11th and 13th weeks, stuffy nose, throat pain and the nasal symptom medication score were significantly relieved in the ‘benifuuki’ containing ginger extract group compared with the placebo group. These results suggested that over one consecutive month, drinking ‘benifuuki’ green tea was useful to reduce some of the symptoms from Japanese cedar pollinosis, and did not affect any normal immune response in subjects with seasonal rhinitis, and the ginger extract enhanced the effect of ‘benifuuki’ green tea.     

Improvement of isolated microspore culture of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) via co-culture with ovary tissues of pepper or wheat

The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.     

Fine-mapping of qRL6.1, a major QTL for root length of rice seedlings grown under a wide range of NH4 + concentrations in hydroponic conditions

Root system development is an important target for improving yield in cereal crops. Active root systems that can take up nutrients more efficiently are essential for enhancing grain yield. In this study, we attempted to identify quantitative trait loci (QTL) involved in root system development by measuring root length of rice seedlings grown in hydroponic culture. Reliable growth conditions for estimating the root length were first established to renew nutrient solutions daily and supply NH₄ ⁺ as a single nitrogen source. Thirty-eight chromosome segment substitution lines derived from a cross between ‘Koshihikari’, a japonica variety, and ‘Kasalath’, an indica variety, were used to detect QTL for seminal root length of seedlings grown in 5 or 500 μM NH₄ ⁺. Eight chromosomal regions were found to be involved in root elongation. Among them, the most effective QTL was detected on a ‘Kasalath’ segment of SL-218, which was localized to the long-arm of chromosome 6. The ‘Kasalath’ allele at this QTL, qRL6.1, greatly promoted root elongation under all NH₄ ⁺ concentrations tested. The genetic effect of this QTL was confirmed by analysis of the near-isogenic line (NIL) qRL6.1. The seminal root length of the NIL was 13.5–21.1% longer than that of ‘Koshihikari’ under different NH₄ ⁺ concentrations. Toward our goal of applying qRL6.1 in a molecular breeding program to enhance rice yield, a candidate genomic region of qRL6.1 was delimited within a 337 kb region in the ‘Nipponbare’ genome by means of progeny testing of F₂ plants/F₃ lines derived from a cross between SL-218 and ‘Koshihikari’.     

Medium,container and genotype all influence in vitro cold storage of apple germplasm

The goal of this study was to evaluate the in vitro storage of apple germplasm by screening a range of genotypes followed by more comprehensive testing of multiple parameters on two genotypes of differing species, Malus domestica cultivar Grushovka Vernenskaya and wild Malus sieversii selection TM-6. Stored plants were rated on a 6 point scale (0 low to 5 high) for plant appearance at 3 month intervals after storage at 4°C. Combinations of carbon source (sucrose and/or mannitol), nitrate nitrogen content (25, 50 or 100%) and plant growth regulators (ABA, BAP, IBA) were studied in three types of containers (tissue culture bags, test tubes or jars). An initial screen of 16 genotypes stored in tissue culture bags indicated that plantlets could be stored at 4°C for 9–14 months without subculture on standard 3% sucrose Murashige and Skoog (1962) (MS) medium with no plant growth regulators (PGRs). In subsequent in-depth studies on the two genotypes, ANOVA indicated highly significant interactions of medium, container and genotype. ‘Grushovka Vernenskaya’ shoots with no PGRs and 3% sucrose remained viable (ratings of ≥1) for 21 months of storage in bags. Storage on reduced nitrogen (MS with 25% nitrogen), PGRs, and 3% sucrose kept ‘Grushovka Vernenskaya’ shoot condition rated >2 at 21 months. Addition of 0.5 or 1 mg⁻¹ abscisic acid (ABA) also improved plant ratings at 21 months. The longest storage for ‘Grushovka Vernenskaya’ was 33–39 months with PGRs and 3% sucrose in either tubes or jars. Addition of abscisic acid (ABA) to the medium did not improve storage of plantlets in jars and tubes at 15 months. TM-6 stored best in tubes on 3% sucrose with PGRs or in jars on 2% mannitol and 2% sucrose. Overall it appears that cold storage of apple shoot cultures can be successful for 21 months in tissue culture bags with 25% MS nitrate nitrogen, 3% sucrose, and no PGRs or for 33 months in jars or tubes on MS with 3% sucrose and PGRs. Preliminary RAPD analysis found no significant differences between plants stored for 39 months and non-stored controls.     

High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis

An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented with 4.65 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23 μM gibberellic acid (GA₃) and 0.46 μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic stability of these regenerant plantlets.     

Non-destructive evaluation of in vitro-stored plants: A comparison of visual and image analysis

Summary In vitro plants, in slow-growth storage require routine evaluation for assessment of viability and need for repropagation. Determination of plantlet health by visual assessment is subjective and varies by genus due to variations in growth pattern and plant structure. Developing a standardized plant evaluation system would improve the efficiency of in vitro storage. This study was initiated to develop digital image analysis techniques for plantlets during slow-growth cold storage and to compare that system with visual examinations. Pear (Pyrus communis L) cultivars were chosen for this initial trial because they have an open structure and clear internode position for image composition. Pear shoots stored at 4°C in tissue culture bags were evaluated monthly by standard visual examination and by digital image analysis. Digital images were evaluated for red, green, blue, modified normalized differences of vegetation index (MNDVI), green/red ratio (G/R), intensity, hue, and saturation at the first two nodes of each, plantlet. At 6 mo., the visual ratings had declined steadily for P. communis ‘Luscious’ and ‘Bartlett—Swiss’, while ‘Belle Lucrative’ and ‘Louse Bonne de Jersey’ ratings did not show significant declines until 9 mo. Correlations between visual ratings and G/R and MNDVI values were significant (r ²>=0.5) for all cultivars. Regression analysis indicated that the MNDVI and G/R ratios changed significantly over the 15-mo. rating period for most cultivars. Intensity, hue and saturation values were not consistently significant and did not correlate with visual ratings. These results will assist in the development of digital imaging as an alternative technique for evaluation of stored in vitro plantlets.     

Screening of a broad range of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) germplasm for <Emphasis Type="Italic">in vitro</Emphasis> rapid plant regeneration and development of an early prediction system

Rice has emerged as a model monocot for studies in agriculture and biotechnology due to its relatively small genome and a ready accessibility to plant material. Tissue culture is one of the tools required for genetic transformation and some breeding programs, and the selection of high-frequency regenerator types is essential for success in these technologies. Thirty-three rice entries with agricultural and biotechnological characteristics of interest were screened with the aim to identify the best regenerators. Entries that exhibited between 50% and 90% regeneration frequencies include ‘Taipei-309,’ ‘Super Dwarf,’ ‘Norin’ (japonica types), PI 312777, ‘Ali Combo’ (indica types), ‘STG-S,’ and ‘LA3’ (red rice types). One third of the entries tested were at least two times better at regeneration than the often-cited regenerator ‘Nipponbare.’ Those entries showing at least 85% frequency of greening or somatic embryo formation at 15 or 30 d on regeneration medium ultimately produced whole plants after 45 d on regeneration medium at high frequency (at least 40%); those entries not reaching the 85% threshold of greening by Days 15 or 30 exhibited moderate (15–40%) to low (less than 10%) frequency of whole plant regeneration. This greening response suggests the means for an early prediction system for identification of useful rice regenerator lines, which would be beneficial for high-throughput screening of germplasm as well as for decreasing the time and cost of in vitro culture.