High polyphenol oxidase activity and low titratable acidity in browning bamboo tissue culture

Summary Tissue browning that frequently results in the early death of bamboo shoots in vitro correlated directly with polyphenol oxidase (PPO, EC 1.10.3.1) activity and inversely with titratable acidity. It was unrelated to the level of endogenous phenols. During the course of culture, timing of PPO activity paralleled that of explant browning. Browning was highest among shoots cultured in a medium of pH 8, which was consistent with the pH optinum of the bamboo enzyme. The pH optimum was first determined with the crude enzyme, then verified with two purified isozymes. Stability of the bamboo PPO was also highest at pH 10. PPO activities of the severely browning Dendrocalamus latiflorus, the moderately browning Phyllostachys nigra, and the relatively non-browning Bambusa oldhamii were inhibited strongly by ascorbic acid, cysteine, sodium diethyldithiocarbamate, and sodium sulfite. But characterization of bamboo PPO according to enzyme inhibitors was not possible because enzyme extracts of the three species gave varied responses to the traditional substances. Nutrient medium addenda of some PPO inhibitors, namely ascorbic acid, cysteine, kojic acid, and thiourea, mainly enhanced browning. However, ferulic acid at 3 mM and lower concentrations reduced the number of brown shoots per culture, although not the percentage of cultures that browned. Polyvinylpyrrolidone failed completely to suppress browning. The two purified isozymes showed different temperature optima for PPO activity: 60°C and 65°C. The purified isozymes displayed a substrate preference for dopamine, or a cathecol oxidase characteristics.     

In vitro regeneration of a mature leguminous liana (Bauhinia vahlii Wight & Arnott)

An in vitro propagation protocol has been developed from mature lianas of Bauhinia valii. Browning was the major obstacle in the establishment of cultures. Explants collected during the growing season (April–June) showed maximum browning; however, browning was minimal during the dormant phase. This problem was circumvented by soaking the sterilized explants in a solution of antioxidant (50 mg l^–1 ascorbic acid+75 mg l^–1 citric acid). The explants were thereafter transferred to culture room conditions after an initial incubation in the dark at 4 °C for 48 h. Shoot proliferation (58%), shoot number (4.5) and shoot length (35 mm) was best in Murashige and Skoog (MS) medium supplemented with 2.5 μM kinetin + 100 mg l^–1 adenine sulfate. Seasonal fluctuations significantly affected the proliferation potential of the explants. March– April was found to be the best season for shoot initiation. Microshoots were rooted on a half-strength, growth regulator-free, agar-gelled Murashige and Skoog medium after a dip in half-strength MS liquid medium containing 1-naph-thaleneacetic acid + indole-3-butyric acid (10 μM). Rooted plantlets were potted and acclimatized under culture room conditions for 4 weeks before transfer to a polyhouse. Received: 9 March 1998 / Revision received: 14 August 1998 / Accepted: 23 September 1998     

Ascorbic acid improves conversion of white spruce somatic embryos

Summary The effects of exogenous applications of ascorbic acid on white spruce somatic embryogenesis were examined. Increasing concentrations of ascorbate (1 μM to 100 μM) in the germination medium enhanced somatic embryo conversion in a linear fashion. At the optimal ascorbate level (100 μM) the number of embryos able to undergo normal conversion, i.e., emergence of both root and shoot, increased from 34% (control) to 58%. The effect of ascorbate had a more pronounced effect on shoot growth than on root emergence; and at 100 μM ascorbate, the percentage of embryos able to produce new leaf primordia increased from 47% (control) to 79%. Root emergence increased slightly from 64% in the control embryos to 74% in the presence of ascorbic acid. The ascorbate-treated embryos were characterized by an enlarged apical region, presumably due to a larger number of leaf primordia produced, and by dark green leaves. When allowed to grow further, these embryos were able to develop into normal plantlets.     

Variability among Vitis vinifera cultivars in micropropagation, organogenesis and antibiotic sensitivity

In vitro culture of 32 Vitis vinifera cultivars and intraspecific hybrids was initiated from axillary buds. The development of roots and shoots was followed during 14 subcultures on two hormone-free micropropagation media. One medium (M64) was used until the 8th subculture, after which it was replaced by G90 medium which was found more suitable for plantlet growth and permitted an increase in the time between subcultures. Large differences in plantlet growth between cultivars were demonstrated on both media. The number of roots had greatest variability between cultivars (CV=39%) as compared with stem length (CV=21-22%) and number of nodes (CV=12-14%). The number of nodes was positively correlated with shoot length whereas root number appeared to be poorly positively correlated with shoot development. Adventitious bud regeneration from leaves was studied for 20 cultivars and averaged 36.7% of regenerative explants with large differences between cultivars (CV=47%). However, organogenic competence was not correlated with micropropagationability. High sensitivity of Vitis vinifera plantlets to kanamycin and hygromycin was demonstrated with a strong interaction between cultivar and antibiotic. At 0.8 mg dm^-3, hygromycin was lethal to plantlets. This effect was only observed at 4 mg dm^-3 for kanamycin, whereas 1 mg dm^-3 stimulated the development of plantlets.Key words: Vitis vinifera, cultivar, micropropagation, antibiotic, organogenesis.      

Alleviation of browning in oak explants by chemical pretreatments

Meristems from 25–90-year-old oak (Quercus robur L. andQ. petraea Matt.) trees and seed embryos were pretreated with polyvinyl pyrrolidone, ascorbic acid, cysteine and citric acid solutions. Tissues were cultured mostly on a WPM medium supplemented with different combinations and concentrations of growth regulators. All the different pretreatments showed a positive effect against the otherwise very rapid and harmful browning of the explants but ascorbic acid (100 mg dm^?3) proved to be the most effective. Shooting was induced from seed embryos and meristems originating from adult trees. Rooted plantlets were obtained from explants of seed embryos.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration in six cultivars of <Emphasis Type="Italic">Capsicum</Emphasis> spp. using different explants

In vitro regeneration from leaf, cotyledon and hypocotyl explants of six cultivars belonging to three species of Capsicum was achieved by direct organogenesis. The cultivar Umorok showed the best response while Meiteimorok, Haomorok, Mashingkha and Uchithi showed intermediate response and the cultivar Chiengpi was the least responsive. Leaf and cotyledon explants regenerated more shoots than hypocotyl explants and the maximum number of shoots were produced on Murashige and Skoog (1962) medium containing 8.8 μM 6-benzylaminopurine (BAP) with 11.4 μM indole-3-acetic acid (IAA). Elongation of shoot buds derived from different explants was achieved on medium containing 2.8 μM IAA and the elongated shoots were rooted on medium containing 2.8 or 5.7 μM IAA and 2.4 or 4.9 μM indole-3-butyric acid (IBA). Four-week old rooted plantlets were hardened and transplanted to the soil. The plantlets showed 90 % survival during transplantation.     

Induction and characterization of in vitro corms of diploid-taro

When in vitro plantlets were cultured in Murashige and Skoog liquid medium supplemented with 8–10% sucrose and 22–44 μM 6-benzylaminopurine, all of the stem explants formed corms. 170–850 μM paclobutrazol increased corm formation, whereas 1700 μM paclobutrazol inhibited corm development. Inclusion of 66 μM 6-benzylaminopurine in 170 μM paclobutrazol treatment resulted in smaller corms, and bigger corms formed in the combination of 1700 μM paclobutrazol and 66 μM 6-benzylaminopurine. No corms formed in 63–630 μM cycocel treatments. In vitro corm growth was also affected by the culture methods. Deep-layer agitated culture yielded corms of up to 2.03 g, with an average fresh weight of 0.7 g, 40 days after induction. In thin layer cultures, corms were up to 1.87 g, with an average fresh weight of 0.5 g. SDS-PAGE analysis of water-soluble proteins revealed changes of polypeptides with corm growth. Compared to smaller ones, corms over 0.2 g had higher dry matter, carbohydrate and anthocyanin content. These corms had a 99–100% survival rate upon transplanting directly to soil after storage at 4 °C for 10 months. This study indicates that the most economic production method of diploid taro seed corm is by thin-layer liquid culture in Murashige and Skoog medium supplemented with 22–44 μM benzylaminopurine and 8–10% sucrose for 6 weeks. The formed corms can be stored at 4 °C up to 10 months and transplanted directly into soil without acclimatization. This revised version was published online in June 2006 with corrections to the Cover Date.     

江西铅山红芽芋脱毒苗球茎愈伤组织诱导及其再生体系的建立

以江西铅山红芽芋脱毒苗为试材,研究不同因素对红芽芋脱毒苗球茎愈伤组织诱导及其再生体系的影响,以期对红芽芋脱毒苗的再生体系进行优化。结果表明,红芽芋脱毒苗球茎愈伤组织诱导的最佳培养基是MS+TDZ 2 mg·L^-1+2,4-D 1 mg·L^-1。红芽芋脱毒苗球茎愈伤组织分化的最佳培养基是MS+TDZ 2 mg·L^-1+NAA 1 mg·L^-1。红芽芋脱毒苗不定芽生根的最佳培养基是1/2MS+NAA 0.5 mg·L^-1+PP₃₃₃ 0.5 mg·L^-1。红芽芋再生苗最好的移栽基质为发酵后的腐锯木屑。红芽芋脱毒苗球茎愈伤组织再生苗移栽时最佳的PP₃₃₃浓度为20～50 mg·L^-1。本试验成功建立了红芽芋脱毒苗球茎愈伤组织的再生体系,为红芽芋脱毒苗转基因的研究和种质创新奠定了基础。     

Direct somatic embryogenesis induced from cotyledons of mango immature zygotic embryos

Summary For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mgl⁻¹ (25μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5mgl⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.     

Effects of sucrose concentration,supporting material and number of air exchanges of the vessel on the growth of in vitro coffee plantlets

Growth of coffee (Coffea arabusta) plantlets cultured in vitroas affected by sugar, types of supporting material and number of air exchanges of the vessel was investigated. Single node cuttings of in vitro coffee plantlets were cultured on half strength MS medium with or without 20 g l⁻¹ sucrose. Two types of supporting material, agar and Florialite, and two levels of air exchange expressed by number of air exchanges per vessel, 0.2 and 2.3 h⁻¹, were studied. At the end of a 40-day culture period, fresh weight, shoot length, root length and leaf area of plantlets when cultured on Florialite soaked in sugar-free medium and under the higher number of air exchanges were greater than those in sugar containing medium. Callus was observed at the shoot base of plantlets grown on agar medium containing sucrose. Photosynthetic ability of coffee plantlets in vitro was also significantly increased when grown on sugar-free medium with the high number of air exchanges and Florialite as a supporting material. This revised version was published online in June 2006 with corrections to the Cover Date.     

Composition of Soluble Nucleotides in Growing Corms of Amorphophallus konjac C. Koch

The nucleotides and the activities of both sucrose synthetase and granular starch synthetase in the konjak corm (Amorphophallus konjac C. Koch) have been investigated as a preliminary experiment on konjak mannan biosynthesis. On chromatographic separation on anion exchange resin and paper of compounds present in the acid ethanol extract from the corms, ascorbic acid, AMP, ADP, ATP, ADP-glucose, UTP, UDP-glucose, GTP, and GDP-mannose were isolated and tentatively identified. An unidentified nucleotide was also isolated.

Of the three nucleotide sugars, UDP-glucose was the most plentiful, while ADP-glucose was the least. The sucrose synthetase in konjak corms was as active as that in other plants. These observations suggest that the mechanism involved in sucrose cleavage in konjak corms is the same as that in other plants, such as sweet potato roots. Starch synthetase bound to starch granules in konjak corms was also found to be active when ADP-glucose was used as glucose donor. But UDP-glucose could not be substituted for ADP-glucose.

Based on these observations the mechanism of konjak mannan biosynthesis is discussed.     

High frequency plant regeneration from protoplasts in cotton <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

A highly reproducible system for efficient plant regeneration from protoplast via somatic embryogenesis was developed in cotton (Gossypium hirsutum L.) cultivar ZDM-3. Embryogenic callus, somatic embryos and suspension culture cells were used as explants. Callus-forming frequency (82.86 %) was obtained in protoplast cultures from suspension culture cells in KM₈P medium with 0.45 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.93 μM kinetin (KIN), 1.5 % glucose and 1.5 % maltose. Protocolonies formed in two months with plating efficiency of 14 %. However, the callus-forming efficiencies from other two explants were low. The calli from protoplast culture were transferred to somatic embryo induction medium and 12.7 % of normal plantlets were obtained on medium contained 3 % maltose or 1 % of each sucrose + maltose + glucose, 2.46 μM indole-3-butyric acid (IBA) and 0.93 μM KIN. Over 100 plantlets were obtained from protoplasts derived from three explants. The regenerated plants were transferred to the soil and the highest survival rate (95 %) was observed in transplanting via a new method.     

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.     

Plantlet production from the male inflorescence tips of <Emphasis Type="Italic">Musa acuminata</Emphasis> cultivars from South India

Inflorescence apices are suitable explants for the rapid in vitro propagation of Musa spp. However, the diploid and triploid banana cultivars showed different in vitro responses with respect to the hormone combinations in Murashige and Skoog medium. The diploid cultivar (Sannachenkadali, AA) induced a maximum number of multiple shoots in 8.9 μM 6-benzyl adenine (BA) whereas the triploid cultivar (Red banana, AAA) exhibited maximum multiplication in 22.2 μM 6-benzyl adenine. MS medium supplemented with 11.4 μM indole acetic acid and 17.8 μM BA was also suitable for shoot proliferation in triploid cultivar but not in the diploid cultivar. The regenerated shoots were rooted in Murashige and Skoog basal medium within 10–15 days. The rooted plantlets were transferred to vermiculite and maintained at a temperature of 25 ± 2°C for 10 days and then at room temperature (30–32°C) for 2 weeks before transferring to potted soil compost mixture. The plantlets showed 100% survival.     

柠檬酸和抗坏血酸对蝴蝶兰叶外植体褐变发生的影响

目的:探究柠檬酸和抗坏血酸对蝴蝶兰叶片外植体褐变发生的影响以及对PPO活性变化影响的作用机理.方法:以褐变率和褐变指数为参考数据,分析柠檬酸和抗坏血酸对外植体PPO活性和PPO反应产物积累的影响以及与外植体褐变发生的关系.结果:分别用100mg/L柠檬酸共培养和50mg/L抗坏血酸浸泡处理叶片外植体,经离体培养3d,褐变率分别比对照降低94.9%和54.9%,离体培养6d,褐变指数低于对照的0.53,分别为0.46和0.36,同时PPO活性降低.结论:推测柠檬酸抑制褐变的原因是直接与酶蛋白作用,抗坏血酸则与新生醌类物质结合.     

Micropropagation of the critically endangered Western Australian species,Symonanthus bancroftii (F. Muell.) L. Haegi (Solanaceae)

A micropropagation protocol was developed for the conservation of the critically endangered Western Australian shrub,Symonanthus bancroftii. It was necessary to screen antioxidant treatments to prevent the occurrence of lethal browning of explants upon excision. Potassium citrate and citric acid (0.1% w/v in a 4:1 ratio) prevented oxidative browning and was superior to the untreated control or other antioxidant treatments tested. Half strength Murashige and Skoog (MS) medium containing 0.5 μM kinetin and 0.25 μM benzyladenine produced three-fold multiplication compared to 1.75×, 1.5×, 1.8× and 1× multiplication for 2.5 μM kinetin + 0.25 μM benzyladenine, 0.5 μM kinetin + 5 μM gibberellic acid, 1 μM kinetin + 3 μM gibberellic acid and half strength MS with no plant growth regulators, over 4 weeks. Root production was achieved with indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) at 0.5/0.5 μM (31% rooting) and 1.0/1.0 μM (36% rooting), after four weeks. Paclobutrazol (PBZ) at 0, 3.4 (1 mg 1⁻¹), 10.2 (3 mg 1⁻¹), or 17 μM (5 mg 1⁻¹) improved tolerance to desiccation after transfer ofin vitro rooted shoots to soil. PBZ at 10.2 μM increased survival to 90% compared to 50% for those plantlets not treated with PBZ. The acclimatisation period from the glasshouse to the shadehouse was 1 week for plantlets treated with PBZ compared to 4 weeks for plantlets without any PBZ. PBZ at 3.4 μM increased the number of roots per shoot compared to untreated controls. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation ofBoswellia serrata Roxb

Anin vitro procedure for large scale multiplication ofBoswellia serrata Roxb. has been developed using cotyledonary node segments. In average 4 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ napthaleneacetic acid (NAA) within 22 d. By repeated subculture technique 90–100 shoots per node could be obtained after 88 d of initial culture. Shoots could be rooted on MS medium containing 1/4 salts, 1% saccharose, and a combination of 0.5 mg dm⁻³ indole-3-butyric acid (IBA) and 0.25 mg dm⁻³ indole-3-acetic acid (IAA). Addition of antioxidants like polyvinylpyrrolidone (PVP-50 mg dm⁻³) and ascorbic acid (100 mg dm⁻³) in both multiplication and rooting media prevented browning of cultures. Approximately 80% of shoots rooted within 8–10 d. Rooted plantlets were kept for 15 d in culture bottles containing Soilrite^TM irrigated with a nutrient solution containing 1/4 MS salts and finally transferred to pots containing soil: Soilrite^TM (1∶1), mixture with 70% transplantation success.     

Sugarcane white leaf phytoplasma in tissue culture: long-term maintenance,transmission, and oxytetracycline remission

Sugarcane white leaf (SCWL)-diseased sugarcane plants collected from Udornthani Province, in north-eastern Thailand, were the source for tissue culture experiments. Explants from axillary buds, meristem tips, and leaves grew optimally in Murashige-Skoog medium containing 0.5 mg/l -naphthaleneacetic acid, 0.5 mg/l 6-benzylaminopurine, and 15% coconut water. Callus development and shoot/root proliferation were more rapid in cultures from diseased than from healthy plants. Disease symptoms continued for 6 years after culture initiation, and SCWL phytoplasma persisted, as confirmed by polymerase chain reaction using both 16S rDNA and 16S-23S rDNA primers. Phytoplasmas in the cultured plantlets were transmissible by grafting to sugarcane and periwinkle, and by feeding of the leafhopper vector Matsumuratettix hiroglyphicus to sugarcane. Although 50% of the plantlets were killed by oxytetracycline at 500 mg/ml, 70–100% of plantlets grown with 200–500 mg/ml oxytetracycline showed symptom remission through 5–8 subcultures. Typical phytoplasma-like bodies, visible by electron microscopy in sieve tubes of untreated diseased plantlets, were absent in antibiotic-treated plantlets. Thus, tissue culture provides a convenient and reliable in vivo system for investigation of SCWL phytoplasma. A preliminary report of this study was presented at the Eighth International Congress of Plant Pathology, Christchurch, New Zealand, 2–7 February 2003     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.     

Regeneration of salvia (<Emphasis Type="Italic">Salvia officinalis</Emphasis> L.) via induction of meristematic callus

Nodular meristematic callus was induced on the basal cut surface of apical shoot explants of salvia cultured on Murashige and Skoog (MS) medium supplemented with 4.5, 13.5, or 22.5 μM thidiazuron (TDZ). Cultures were incubated in the dark for 1 wk and then transferred to light conditions for 4 wk. A higher percentage of explants developing callus was observed on medium containing either 4.5 or 13.5 μM TDZ, although explants on 4.5 μM developed larger calluses. The callus was maintained on medium containing 4.5 μM TDZ and 0.45 mM ascorbic acid. Shoot differentiation, after each of three successive maintenance passages, was induced from callus grown on medium containing either 4.4 or 8.8 μM benzyladenine (BA). A greater number of shoots were harvested from callus differentiated on BA (4.4 or 8.8 μM) medium with 0.45 mM ascorbic acid added. Shoots developed roots on MS medium supplemented with 4.9 μM of indole-3-butyric acid. The addition of ascorbic acid to the shoot differentiation medium enhanced rooting, number of roots per shoot, and survival rate. Approximately 75% in vitro plantlets were acclimatized to ex vitro conditions. Histological investigations confirmed both adventitious meristem initiation during the callus induction phase, and subsequent organogenic shoot development on the differentiation medium. The novel protocol for the meristematic callus induction and plant regeneration in this study may be useful for biotechnological applications for salvia improvement via genetic transformation or mutagenesis and in vitro propagation approaches.