Volume 6 Number 1 2012

CONTENTS AND ABSTRACTS

Aya Hatano-Iwasaki, Ken’ichi Ogawa (Japan) Biomass Production Is Promoted by Increasing an Aldolase Undergoing Glutathionylation in Arabidopsis thaliana (pp 1-8)

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Original Research Paper: We previously identified a putative fructose-1,6-bisphosphate aldolase (FBA), which was designated as FBA1 (At2g01140.1), as a protein undergoing glutathionylation in Arabidopsis. Here we show that increasing FBA1 activity can improve biomass production by enhancing leaf area-based CO₂ assimilation. Transgenic Arabidopsis plants with increased accumulation of FBA1 protein (35S-FBA1 plants) exhibited enhanced photosynthetic CO₂ assimilation rate with increased FBA activity, and yielded more seeds and aerial biomass than wild-type plants under nitrogen nutrient conditions suitable for biomass production. FBA activity and photosynthetic CO₂ assimilation rate decreased in glutathione-deficient mutants, cad2-1 and pad2-1, with decreased glutathione levels, while FBA protein levels were little affected in the mutant plants. An in vitro enzyme assay for FBA from mutant and wild-type plants revealed that FBA activity can be increased and recovered by 2.5 mM glutathione, but not by a strong reducing agent, 20 mM dithiothreitol. We found a positive correlation between photosynthetic CO₂ assimilation rate and FBA activity among plants with various levels of FBA protein and of glutathione, and this correlation was strengthened (r²=0.955) with increasing light intensity from 25 to 1000 µE m^-2 s^-1. On the other hand, there was much less correlation between photosynthetic CO₂ assimilation rate and Rubisco protein (r²=0.013) or total activity (r²=0.005), which have been generally considered to be a major limiting factor of photosynthesis. These results indicate that FBA activity is a limiting factor for photosynthetic CO₂ assimilation and biomass production, even at the light intensity at which Rubisco is fully activated, and that it is likely to be regulated by glutathione.

Hussein Alzubi (Syria), Luz Marcela Yepes, Marc Fuchs (USA) Enhanced Micropropagation and Establishment of Grapevine Rootstock Genotypes (pp 9-14)

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Original Research Paper: The effect of medium composition, antioxidant and adsorbent of polyphenolic compounds on micropropagation of grapevine rootstocks 3309 Couderc, 110 Richter, 101-14 Millardet et De Grasset, Vitis riparia Gloire de Montpellier and Teleki 5C was determined. Shoot length, leaf number and size, as well as root number, weight and length were consistently higher on woody plant medium (WPM) (Lloyd and McCown 1981) relative to full strength or half-strength Murashige and Skoog (MS) medium (Murashige and Skoog 1962) and Martin et al. medium (MM) (Martin et al. 1987). Supplementing WPM with 37 mg l^-1 cysteine as antioxidant promoted better rooting and enhanced plant development for most rootstock genotypes. The presence of cysteine in WPM also provided homogeneous growth, regardless of the position of nodal segments on the initial subcultured plants, likely by breaking the apical meristem dominance inhibitory effect on axillary bud proliferation. Our micropropagation protocol facilitated a fast and uniform multiplication of grapevine rootstocks in tissue culture and a successful transfer and growth of micropropagated plants in the greenhouse.

Vinay Kumar, Sudesh Kumar Yadav (India) Developmental Effect on Transcript Expression of Genes Encoding Enzymes for Flavan-3-ols Synthesis and its Content in Leaves and Flowers of Tea (Camellia sinensis (L.) O. Kuntze) (pp 15-20)

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Original Research Paper: The production of monomeric flavan-3-ols mainly comprises of catechin and epicatechin, epigallocatechin and epicatechingallate, and their polymeric form as proanthocyanidins is highly controlled during the developmental stages of leaf and flower. We studied the accumulation of monomeric flavan-3-ols, anthocyanins and proanthocyanidins during the developmental stages of leaf and flower in relation to the expression of flavan-3-ols-specificgenes of tea (Camellia sinensis). The expression analyses of flavan-3-ols-specific genes were also studied in the presence of exogenous monomeric flavan-3-ols such as catechin and epicatechin during different development stages of both organs. We selected two developmental stages of leaf (leaf bud (LB) and old leaf (OL)) and three stages of flower development (flower bud (FB), semi-mature flower (SMF) and fully mature flower (FMF)) to assess various activities. The monomeric flavan-3-ols were highest in LB and FB and lowest in OL and FMF. The pattern of accumulation of anthocyanins was opposite to monomeric flavan-3-ols content during different development stages of the leaf and flower. In leaves, proanthocyanidins, oligomeric and/or polymeric forms of flavan-3-ols were higher in OL than in LB while in flowers, proanthocyanidins were lowest in FMF. Expression of the DFR gene encoding dihyroflavonol 4-reductase, the LAR gene encoding leucoanthocyanidin reductase and the ANR gene encoding anthocyanidin reductase was correlated with the accumulation of monomeric flavan-3-ols during leaf and flower development. Expression of the ANS gene, encoding anthocyanidin synthase, was only detected in OL, which also contained the highest anthocyanin content. Exogenous exposure of flavan-3-ols (catechin and epicatechin) modulated the expression of DFR, LAR and ANR in OL and LB tissues but only modulated the expression of LAR during all stages of flower development. This study provides an overview of new insight for monomeric flavan-3-ols biosynthesis, and their transcript regulation during leaf and flower development of C. sinensis.

Soumya V. Menon, T. V. Ramana Rao (India) Enzyme Activities during the Development and Ripening of Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) Fruit (pp 21-26)

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Original Research Paper: Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai; Cucurbitaceae), is an excellent source of sugars and antioxidants, mainly lycopene, which makes it a nutritionally good fruit. The present study was carried out with the purpose of evaluating the activities of various enzymes in relation to the biochemical processes occurring at five sequential stages of watermelon fruit: young, pre-mature, mature, pre-ripened and ripened. Maximum activity (0.010 mg maltose released/mg protein) of β-amylase (free), one of the starch hydrolyzing enzymes, was found in the pre-mature stage, while β-amylase (bound) exhibited highest activity (0.063 mg maltose released/mg protein) during the pre-ripened stage. Sugar metabolizing enzymes in the relevant metabolic pathways of sugars were also quantified. Sucrose phosphate synthase and sucrose synthase exhibited maximum activities in the young and pre-mature stages, respectively. These activities were significantly higher (P < 0.05) than in other development and ripening stages. The sucrose-cleaving enzyme, acid invertase, showed remarkably high activity (0.014 µmol/h/mg protein) in the ripe stage, as did neutral invertase in the mature stage (0.029 µmol/h/mg protein). β-Galactosidase activity (0.035 µmol pnp/mg protein) was maximum in the mature stage, while activities of polygalacturonase (0.025 mg glucose released/mg protein) and cellulase (0.01 mg glucose released/mg protein) were maximum in the young stage. The specific activity of peroxidase was significantly high (0.0034 units/mg protein) in the pre-mature stage whereas polyphenol oxidase exhibited inconsistent activity, but with a maximum in the ripened stage (3.49 units/mg protein). The overall results indicate a positive relation between enzymes (sucrose phosphate synthase, sucrose synthase, β-galactosidase, amylases and polyphenol oxidase) and the development and ripening of watermelon fruit.

Tukaram Dayaram Nikam, Jitendra Gopichand Patil, Mahendra Laxman Ahire, Savaliram Goga Ghane, Kirti Manik Nitnaware, Vikas Bandu Naikawadi (India) Axillary Multiplication of Ceropegia mahabalei Hemadri & Ansari and Ceropegia media (Huber) Ansari: Critically Endangered Ethnomedicinal Herbs of Western Ghats, Maharashtra State of India (pp 27-33)

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Original Research Paper: A rapid micropropagation system was developed for critically endangered ethnomedicinal herbs Ceropegia mahabalei Hemadri & Ansari and C. media (Huber) Ansari. For shoot multiplication nodal, internodal and leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of cytokinins [(6-Benzyladenine: BA or Kinetin: Kin (0.0-10.0 µM)] alone and in combination with auxins [(α-naphthaleneacetic acid: NAA; indole-3-acetic acid: IAA; 2,4-dichlorophenoxyacetic acid: 2,4-D (0.5-1.5 µM)]. Maximum number of shoots were produced on subculture of nodal explants of C. mahabalei and C. media on MS medium supplemented with 5.00 µM BA. The type and age of explant and addition of auxins (NAA, IAA or 2,4-D: 0.5-1.5 µM) in the medium influenced shoot multiplication. The best medium for rooting of shoots of both the species was liquid MS medium containing 1.0 µM NAA and 4% sucrose. Well rooted plantlets were transplanted into soil and about 88% of the plantlets survived well upon transferred to natural conditions. The plantlets were morphologically identical to the parental plants. This work may be helpful for in vitro propagation, ex situ conservation and genetic manipulation of these species.

Mahendra Laxman Ahire, Pradip Pandit Patil, Polavarapu B. Kavi Kishor, Tukaram Dayaram Nikam (India) Micropropagation and Assessment of Antibiotic Selection in Vitro of Bacopa monnieri (L.) Pennell (pp 34-39)

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Original Research Paper: Bacopa monnieri (L.) Pennell (Brahmi), Scrophulariaceae, is one of the sources of medhya rasayan drugs (that counteract stress and improves intelligence and memory) of Ayurveda. The aim of the present study was to assess tissue culture conditions and antibiotic selection which could be useful for genetic transformation of this important medicinal plant. An efficient, simple and reproducible system for plant regeneration through leaf and nodal explants was devised. Sensitivity of the nodal explants against cefotaxime (Cef), kanamycin (Kan) and hygromycin (Hyg) was established. Leaf and nodal explants produced about 90 shoots on Murashige and Skoog (MS) medium fortified with 1.0 mg/L 6-benzyladenine (BA) after 28 days of incubation. Subculture to fresh MS medium resulted in shoot elongation; the shoots obtained grew well and were healthy. The transfer of shoots to liquid MS medium supplemented with 0.2 mg/L α-naphthaleneacetic acid (NAA) resulted in 100% rooting. The rooted shoots, on transfer to plastic containers containing a mixture of garden soil + sand (1:1), could acclimatize within 14 days under glasshouse conditions. The acclimatized in vitro-grown plants showed 100% survival after transfer to soil in earthen pots. A threshold limit of survival of nodal explants was observed at 500 mg/L Cef, 100 mg/L Kan and 15 mg/L Hyg.

Shiwali Sharma, Anwar Shahzad (India), Mohd. Shahid (Kingdom of Bahrain/India), Noor Jahan (India) An Efficient in Vitro Production of Shoots from Shoot Tips and Antifungal Activity of Spilanthes acmella (L.) Murr. (pp 40-45)

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Original Research Paper: An efficient method for the propagation of Spilanthes acmella (L.) Murr. through shoot tips (collected from 3 week-old axenic seedlings) has been successfully developed. This protocol can be employed on a commercial scale for the production of spilanthol. Among the cytokinins, 6-benzyladenine (BA), kinetin (Kn) and 2-isopentenylaminopurine (2-iP) were tested. Murashige and Skoog (MS) medium supplemented with 1.0 µM BA was optimum for inducing bud break. When auxin was augmented with an optimal cytokinin concentration, the regeneration efficiency of explants was enhanced. The maximum response (96%) with highest number of shoots per explant (i.e., 33.0) was possible on MS medium containing 1.0 µM BA and 0.1 µM α-naphthaleneacetic acid (NAA). Small shoots (3.0-4.0 cm) were rooted in vitro with half-strength MS medium containing 2.5 µM NAA, forming a maximum of 32.2 roots/shoot. The well developed micropropagated plants were successfully acclimatized within 4 weeks in Soilrite^TM and planted ex vitro in garden soil, farmyard soil and sand (2: 1: 1), where they grew well without any apparent morphological variation from the parent plant. Moreover, the phytomedicinal effect of various plant tissues was also evaluated against human pathogenic fungi. The alcoholic extracts of in vitro plant were more effective than in vivo plant materials. Maximum inhibition zone (MIZ) was noticed against Candida krusei followed by Candida albicans, Aspergillus fumigatus and Aspergillus niger. Among different explants, flower heads showed best response against C. krusei as highest of 12.3 and 12.0 cm MIZs were noticed for in vitro and in vivo source, respectively.

Budi Winarto (Indonesia), Jaime A. Teixeira da Silva (Japan) Sterilization Procedure for in Vitro Culture of Leatherleaf Fern (Rumohra adiantiformis) (pp 46-50)

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Original Research Paper: When explants are introduced to the in vitro environment, sterilization is a fundamental process allowing contamination to be eliminated. A new sterilization procedure for a novel in vitro propagation of (Rumohra adiantiformis) was successfully devised using rhizomes – which are typically heavily contaminated – as the donor explant. The most effective sterilization procedure for in vitro micropropagation involved pretreatment of rhizomes in 80% alcohol for 3 min, washing them under tap water for 3 h then immersing them in 0.05% mercuric chloride for 10 min, followed by 96% alcohol for 1 min. Finally explants were rinsed 6 times (5 min each rinse) using sterile distilled water. This combination of treatments reduced the percentage of contamination to 33% and stimulated the percentage of rhizome regeneration to 73% with 2.3 rhizomes regenerated/replication (= 5 ex vitro rhizomes). This in vitro sterilization procedure will have an effective impact on the establishment of explants in vitro and on the micropropagation of leatherleaf fern.

XiaoNan Yu, HongJuan Wu, Tong Pan (China), Jaime A. Teixeira da Silva (Japan) Multiple Shoot and Callus Induction of Herbaceous Peony (Paeonia lactiflora Pall.) (pp 51-56)

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Original Research Paper: Underground buds of herbaceous peony (Paeonia lactiflora Pall.) ‘Zhong Sheng Fen’ were used as explants for axillary shoot induction while stems, petioles and leaves of ‘Da Fu Gui’ and ‘Tao Hua Fei Xue’ were used as explants for callus induction. The effects of different basal media and concentrations of plant growth regulators (PGRs) on induction were investigated to establish an aseptic regeneration system. The best medium to induce and proliferate shoots was modified half-strength Murashige and Skoog (MS) medium with double the concentration of Ca²⁺ supplemented with 1 mg l^-1 gibberellic acid (GA₃) plus 1 mg l^-1 6-benzylaminopurine (6-BA). Two successive steps were adopted for rooting shoots. Shoots were first cultured on Woody Plant Medium (WPM) plus 0.5 mg l^-1 1-naphthyleneacetic acid (NAA) for 10-15 days in the dark, then shoots were transferred to PGR-free WPM medium containing 0.2% activated charcoal; in this case, rooting could reach 50%. The best explants for callus induction were young stems, and the best basal medium for callus induction was WPM for ‘Da Fu Gui’ and ‘Tao Hua Fei Xue’.

Syed Arshad Hussain (India), Jaime A. Teixeira da Silva (Japan), Azra Nahaid Kamili, Ali Mohammad Shah (India) In Vitro Propagation of Parrotiopsis jacquemontiana (Decne) Rehd. Using Mature Tree Explants (pp 57-60)

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Original Research Paper: The vegetative propagation of Parrotiopsis jacquemontiana (Decne) Rehd., an endemic species of the Hamamelidaceae family growing wild in Kashmir, is still considered to be difficult owing to its difficult-to-root stem cuttings. The present study reports, for the first time, an in vitro propagation technique for this species using shoot apex and nodal stem segments of a mature tree as explants. The initial shoot cultures were established in agarified Murashige and Skoog (1962) basal medium supplemented with a cytokinin (6-benzylaminopurine (BA) or kinetin at 1-10 µM). The explants secreted a large amount of phenolic substances which frequently led to tissue browning; this problem could be overcome by washing the explants for at least 30 min before inoculation and frequently transferring them to fresh medium during the culture establishment phase. The initial shoots were subcultured in a multiplication medium having a combination of an auxin (1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA) at 2 µM) and a cytokinin (BA) at 2.5-10 µM. Shoot multiplication was best (5-6 shoots/culture) on MS medium having BA and IAA at 5 and 2 µM, respectively. In vitro shoots 2.5 cm or longer were successfully rooted in auxin-supplemented ½-MS medium within 8 weeks. IAA, NAA or IBA at 2.5 and 5 µM were the most effective concentrations for inducing rooting. The plantlets were acclimatized with 40% survival.

XiaoNan Yu, LiHui Wang (China), Jaime A. Teixeira da Silva (Japan) Change of Endogenous Hormones Inside Paeonia lactiflora Buds during Winter Dormancy (pp 61-63)

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Short Communication: Variation in and the ratios of endogenous hormones in the terminal buds of 4-year old traditional Chinese peony cv. ‘Da Fu Gui’ throughout the dormant period were studied by HPLC to attempt to unravel the mechanism of winter bud dormancy in this woody ornamental. Abscisic acid (ABA) content began to increase on November 20 and peaked on December 10 while gibberellic acid (GA₃) content started to increase on December 10 and dropped off by December 30 while the trend for cytokinin (CK) was similar to that of GA₃. The level of indole-3-acetic acid changed little throughout the period of dormancy. GA₃/ABA and CK/ABA values dropped as dormancy progressed and rose as dormancy was released.

Vishnu Sukumari Nath, Muthukrishnan Senthil alias Sankar, Vinayaka Mahabaleswar Hegde, Muthulekshmi Lajapathy Jeeva, Raj Shekar Misra, Syamala Swayamvaran Veena (India) A Simple and Efficient Protocol for Rapid Regeneration and Propagation of Taro (Colocasia esculenta (L.) Schott.) in Vitro from Apical Meristems (pp 64-66)

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Short Communication: An efficient and simple protocol was developed for the in vitro regeneration and propagation of taro (Colocasia esculenta) cv. ‘Muktakeshi’. Apical meristems (~1 cm) excised from leaf blight-resistant taro cultivar cv. ‘Muktakeshi’ grown in a net house were used as explants. Most multiple shoots (i.e., 3.6/explant), which formed on Murashige and Skoog (MS) medium supplemented with 5 mg L^-1 6-benzyladenine and 1 mg L^-1 α-naphthaleneacetic acid, could be rooted by transferring the 4-week-old shoots to MS basal medium without plant growth regulators. After 2 weeks, well developed plantlets were hardened in plastic cups in potting mixture (vermiculite + sand, 1:1, v/v). Acclimatized plants were transferred to 30-cm plastic pots containing top soil and vermicompost (3:1, v/v) where they grew well.

Anu Bhatt, Shubhi Kansal, Rajinder Singh Chauhan, Hemant Sood (India) Modified Tissue Culture Procedures for Micropropagation of Apple Root Stocks (pp 67-72)

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Research Note: The low yield of apples (Malus spp.) can be amounted to the uncertainties of the monsoon, dependence over the old cultivars and prone to pathogen infestation. This warrants the development of a cost-effective micropropagation technology for the rapid multiplication of commercially important root stocks which can be utilized for re-plantation in apple orchards. Axillary shoot tips of M9 rootstock formed most multiple shoots on Murashige and Skoog (MS) medium supplemented with 3 mg/l 6-benzyladenine (BA), 2 mg/l kinetin (KN), 3% (w/v) sucrose and 0.8% (w/v) agar-agar, with 85.7% of shoot apices forming multiple shoots. To reduce the cost of media components for commercial production of the planting material from root stocks, sucrose was replaced with table sugar and agar-agar was completely omitted. The low-cost medium combination of MS liquid medium supplemented with 2 mg/l BA, 3 mg/l KN and 3% (w/v) table sugar was best, resulting in 22 shoots/explants. There were no significant differences in multiple shoot formation/explant, relative growth and vigor of shoots and frequency of root formation in shoots independent of whether medium contained sucrose or table sugar. In vitro regenerated plantlets were successfully hardened and transferred to the field. The substitution of sucrose for table sugar and the omission of agar-agar from the medium reduced the cost/liter medium by approximately 80-fold when other cost-effective alternatives such as tap water and autoclavable polybags were used, assisting in easier and more effective commercialization.