Rapid plant regeneration protocol for cluster bean (Cyamopsis tetragonoloba L. Taub.)

Summary

An efficient in vitro regeneration procedure using thidiazuron (TDZ) has been developed to allow high frequency, multiple shoot induction from cotyledonary node explants of cluster bean (Cyamopsis tetragonoloba). Shoot bud induction occurred on Murashige and Skoog (MS) medium after 4 weeks in the presence of TDZ, followed by transfer onto shoot multiplication and elongation media containing MS salts, B5 vitamins, and different combinations of auxins and cytokinins. Multiple shoots were induced at all levels of TDZ in the medium, but the best proliferation capacity occurred at 5 µM TDZ. Combinations of auxins and cytokinins showed a stimulatory effect on shoot multiplication and also on the length of the newly formed shoots. Maximum shoot induction [i.e., the highest number of shoots (16.0 ± 0.94) per explant] was obtained on agar-solidified medium containing 5 µM benzyladenine (BA) with 0.5 µM indole-3-acetic acid (IAA). Rooting of in vitro-regenerated shoots was achieved in ex vitro conditions by a pulse treatment with 300 µM indole-3-butyric acid (IBA) for 15 min. Rooted plantlets were transferred to soil where 70 – 75% attained sexual maturity and produced viable seeds under greenhouse conditions. The present regeneration system is efficient and can be used in various in vitro manipulation studies.     

In vitro organogenesis of Abutilon indicum (L.) Sweet from leaf derived callus and assessment of genetic fidelity using ISSR markers

This study reports on in vitro regeneration of Abutilon indicum plantlets through callus mediated organogenesis. The leaf explants implanted on Murashige and Skoogs (MS) medium supplemented with 4.52 µM 2, 4-Dicholorophenoxy acetic acid (2,4-D) and 8.88 µM 6 Benzyladenine (BA) showed highest response (70.3%) for callus proliferation, but these callus did not showed any morphogenetic differentiation on the same medium even after 12 weeks. Whereas, subsequent sub-culture of this green proliferated callus on MS medium added with 2.68µM α-Napthalene acetic acid (NAA), 8.88µM BA and 543 µM Adenine sulphate showed the highest frequency (62.2%) of multiple shoot-buds production and also elongation of shoots. Well developed shoots were efficiently rooted in vitro on half strength MS medium supplemented with 7.38 µM Indole-3-butyric acid (IBA). Seventy per cent of in vitro regenerated plantlets were successfully established in garden and were morphologically alike to the donor plants. The genetic homogeneity of these in vitro regenerated plantlets was also affirmed by inter simple sequence repeat (ISSR) analysis using eight ISSR primers. This standardised in vitro organogenesis protocol supplements a good platform for the conservation of A. indicum germplasms and also caters for the needs of the herbal industry.     

Shoot tip culture in mango: Influence of medium,genotype, explant factors,season and decontamination treatments on phenolic exudation,explant survival and axenic culture establishment

Summary

Attempts to establish shoot tip culture in mango (Mangifera indica L.) genotypes ‘Alphonso’, ‘Totapuri’, ‘Banganapalli’ and ‘Arka Anmol’ indicated that the problems of phenolic exudation, medium discoloration and explant browning were interrelated and influenced by a number of factors including medium, genotype, explant, season and decontamination treatments. Less phenolic exudation and better explant survival were observed in ½MS medium than full MS and in semi-solid than in liquid medium. Among explant factors, age and shading of shoots, length, thickness and scaling injuries on explant and lower cut petioles coming in contact with medium were important, but not the age of tree and the number of leaves on the shoot. Use of charcoal was beneficial but other adsorbents, antioxidants, dark incubation and submerged culture were not advantageous. The effect of season was prominent with least phenolics, minimum microbial contamination, best explant survival and genotype dependent growth response in current season’s semimature shoots collected during June-August. Deep seated microbial contaminants could not be checked completely. A scheme for establishing in vitro culture is suggested.     

Clonal propagation of Poncirus trifoliate through culture of shoot primordia

Summary

In Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.     

Adventitious shoot regeneration from leaf segments of in vitro cultured shoots of the apple rootstock Jork 9

SUMMARY

Adventitious shoot formation was investigated using leaf segments of in vitro cultured shoots of the apple rootstock Jork 9. Regeneration capacity was influenced by the pretreatment of the mother shoots, macroelements, hormone concentrations, the gelling agent and the carbohydrate source. The highest regeneration rate and most shoots per leaf explant resulted from young leaves on a medium based on MS macroelements supplemented with 22 µM BAP and 0.1 µM_NAA together with sorbitol, at concentrations of 165 mM or 220 mM. Sorbitol was more effective than sucrose, glucose, fructose or a combination of these sugars. A cold and dark pretreatment of the shoots enhanced the formation of adventitious shoots.     

In vitro propagation of GF677 hybrid rootstock (Prunus persica × Prunus amygdalus) from mature cotyledons

Adventitious shoot regeneration from mature cotyledons of GF677 rootstock (Prunus persica × Prunus amygdalus) was achieved in vitro. Thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-yl-urea; TDZ) at 32 µM gave the highest percentage of cotyledons forming adventitious shoots (68.8%) and the highest number of shoots per cotyledon (4.8) on Quoirin and Lepoivre (QL) medium. On QL medium containing 32 µM TDZ, exposure of the proximal segments of cotyledons to darkness at the start of culture increased the percentage of cotyledons forming adventitious shoots (62.5%) when compared with those kept under light conditions (15%). A combination of 0.72 µM gibberellic acid and dark treatment resulted in at least 2.7-fold more elongated shoots than non-treated shoots. The highest rooting percentage (100%) occurred on 0.5× Murashige and Skoog medium supplemented with Gamborg (B5) vitamins and 2 mg l^?1 indole-3-butyric acid. Rooted plantlets were acclimatised under greenhouse conditions with a 70% survival rate.     

Micropropagation of bay laurel (Daphne gnidium L.)

Summary

Procedures have been developed for the micropropagation of Daphne gnidium, a shrub species of ecological interest, using explants of juvenile and adult origin. Shoot proliferation rates were significantly affected by both salt formulation and benzylade- nine concentration. Best results were obtained on WP medium with 5 µM BA. The presence of indoleacetic acid in the induction medium improved BA-induced axillary bud proliferation from juvenile explants. Rooting of shoots produced in culture was difficult, especially those of adult origin. Besides an absolute requirement for auxin, calcium concentration and the pH of the medium affected the formation of adventitious roots from regenerated shoots of D. gnidium.     

弯刺蔷薇与大花白木香越冬抗寒性及其生理差异分析

以弯刺蔷薇(Rosa beggeriana)和大花白木香(R.fortuneana)当年生叶片和枝条为试材,采用电解质渗出率拟合Logistic方程确定低温半致死温度(LT_(50))评价抗寒性,采用过碘酸雪夫氏染色观察茎中淀粉积累,测定相对含水量、丙二醛、可溶性糖、脯氨酸、脱落酸含量以及超氧化物歧化酶、过氧化物酶和过氧化氢酶活性,研究其越冬期间抗寒性变化及其生理基础。结果发现:两种蔷薇叶片抗寒能力相当,但弯刺蔷薇枝条抗寒性远强于大花白木香。与大花白木香相比,弯刺蔷薇在低温驯化初期累积了更多的可溶性糖,并在叶片脱落前可能通过增加的ABA信号促进可溶性糖由叶片向茎中运输并贮存为淀粉,在严冬时期淀粉水解形成更多的渗透调节物质,并通过更高的SOD活性维持氧化还原平衡;而大花白木香中只发现了比弯刺蔷薇中更高的脯氨酸含量和POD活性,这可能难以维持其渗透压和氧化还原平衡并造成了更多的膜脂过氧化伤害,最终导致其枝条的抗寒性远小于弯刺蔷薇。     

Colchicine- and trifluralin-mediated polyploidization of Rosa multiflora Thunb. var. inermis and Rosa roxburghii f. normalis

Rosa multiflora Thunb. var. inermis and Rosa roxburghii f. normalis are two important diploid species for rose breeding. Interspecific crosses between diploid species and tetraploid rose cultivars result in triploids with generally decreased fertility. A most promising gene transfer strategy is the production of fertile tetraploid plants before mating with tetraploids. In our study, germinating seeds of R. multiflora and R. roxburghii were treated with colchicine, and cotyledon-stage seedlings of R. multiflora were treated with colchicine or trifluralin in order to obtain tetraploid plants. Also, various concentrations of antimitotic agents and different treatment durations were tested. The results showed the antimitotic agents affected the plant morphological characteristics (i.e. plant height, stem diameter) of R. multiflora and R. roxburghii. Twenty-three tetraploid plants were obtained from the germinating seed treatments (21 from R. multiflora and two from R. roxburghii), but no tetraploid plants were obtained from the seedling treatments. The highest rate of chromosome doubling (25.0%) was achieved when germinating seeds of R. multiflora were treated with 0.2% colchicine for 12 h. At the same time, marked morphological variation was observed and analyzed among tetraploid plants and their corresponding diploids of R. multiflora.     

The Influence of Some Genetic and Environmental Factors on the Concentration of L-Ascorbic Acid in the Tomato Fruit

Summary

An efficient, reproducible protocol has been developed for in vitro multiplication of Sida cordifolia L. High-frequency, multiple shoots (90%) were obtained indirectly from nodal explants. Callus was induced when nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg l^–1 Kinetin (Kn). These nodal calli were then cultured in order to differentiate multiple shoots on MS medium supplemented with 0.5 mg l^–1 Kn plus 0.5 mg l^–1 naphthaleneacetic acid (NAA). Roots were induced from these multiple shoots following culture on MS medium supplemented with 0.8 mg l^–1 NAA for 4 weeks. Finally, these in vitro plantlets were hardened, acclimatised, and successfully transferred to the field.     

Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.     

Micropropagation of Prunus scoparia,a Suitable Rootstock for Almond under Drought Conditions

Prunus scoparia is a wild deciduous shrub, usually living on dry calcareous soils of the rocky mountains and has been used as a grafting rootstock for domesticated almonds to provide drought resistance. In the current study, micropropagation ability of P. scoparia was investigated using cytokinin and auxin. Uniform nodal shoot pieces (3–5 cm in length) of seedlings were used as explants. The explants were disinfected with 10% sodium hypochlorite solution. For adventitious shoot induction and proliferation, Murashige and Skoog (MS) media containing 7.00 g/l agar and 30.00 g/l sucrose containing five concentrations of benzyl adenine (BA) (0.00, 0.50, 1.00, 2.00, and 4.00 mg/1) and also containing six concentrations of Thidiazuron (TDZ) (0.00, 0.50, 1.00, 2.00, 5.00, and 7.00 mg/1) were compared. For rooting, in vitro shoots (2–3 cm) were transferred into ½ MS medium supplemented with 30 g/l sucrose, 7.50 g/l agar, and different concentrations of IBA (0.00, 0.25, 0.50, and 1.00 mg/l) and NAA (0.00, 0.25, 0.50, and 1.00 mg/l). Based on the results obtained for shoot proliferation, only 2.00 and 4.00 mg/l BA and 2.00 mg/l TDZ concentrations generated shoots, while other treatments did not show shoot proliferation. Among the three treatments that generated shoots, the best results for shoot number, leaf number, and leaf color quality were observed in media containing 2.00 mg/l TDZ. Based on the results obtained for rooting, the effect of IBA concentrations on the rooting percentage, root number, and root length was significant. Among IBA concentrations, only 0.50 mg/l IBA induced rooting, while there was no rooting in the media containing other IBA concentrations. None of the NAA concentrations showed rooting. In conclusion, MS culture medium supplemented with 2.00 mg/l TDZ and ½ MS culture medium supplemented with 0.50 mg/l IBA are suggested for in vitro shoot proliferation and rooting of P. scoparia, respectively. The results presented herein could be used for in vitro selection and micropropagation of P. scoparia.     

Role of sucrose synthase and invertases during petal senescence in rose (Rosa hybrida L.)

Summary

The roles of sucrose synthase and invertases were explored in relation to petal senescence in rose (Rosa hybrida L.). A developmental shift in the activities of these enzymes was observed. Higher sucrose synthase activity (0.52 – 0.95 µmol sucrose min^–1 mg^–1 protein) was observed during the initial stages (S1 and S2) of flower bud development, in contrast to invertases. However, the lower activity (0.56 µmol sucrose min^–1 mg^–1 protein) of sucrose synthase at a later stage (S6) of senescence could help the mobilisation of vacuolar sucrose. The different isoforms of invertase exhibited variable levels of activity. Insoluble acid invertases (IAI) were the most active (11.01 µmol glucose min^–1 mg^–1 protein), followed by soluble acid invertases (SAI; 8.02 µmol glucose min^–1 mg^–1 protein), and soluble neutral invertases (SNI; 0.74 µmol glucose min^–1 mg^–1 protein) at Stage-4. A significant decline in invertase activities (IAI, 0.98; SAI, 1.25; SNI, 0.32 µmol glucose min^–1 mg^–1 protein) coincided with higher levels of ethylene production at the later stages (S5 and S6) of flower bud development and senescence. We propose that developmental as well as ethylene-mediated pathways account for petal senescence in rose.     

植物生长调节剂对线艺春兰根状茎的增殖与分化的影响

 以线艺春兰‘绿云’的类原球茎为材料, 研究了植物生长调节剂等因素对根状茎的增殖与分化的影响。结果表明: 长期继代培养及保存宜选用1 /2MS固体培养基。细胞分裂素6-BA浓度为1.0 mg·L ^{- 1}时根状茎的增殖效果最佳。0～3.0 mg·L ^{- 1}范围内IBA促进根状茎的生长较NAA效果好。1/2MS + 6-BA 1.0 mg·L ^{- 1} + IBA 1.0 mg·L ^{- 1}培养基最利于根状茎的分化, 芽分化率达到36.9%。培养基添加芋艿糊可显著促进根状茎的增殖与生长。根状茎分割以掰开方式且接种团块带有3～5条根状茎较适宜。线艺春兰叶片上的腹轮艺通过组培可以稳定遗传。     

Two-way germination system of encapsulated clonal propagules of Vitex trifolia L.: an important medicinal plant

In the present study we have developed an efficient and effective method of synthetic seed production and its two-way germination system of Vitex trifolia, for easy transport of the propagules and efficient utilization of its in vitro regeneration system. Nodal segments harvested from 8-week-old in vitro cultures were encapsulated in calcium alginate beads. Three percent (w/v) Na-alginate polymerized in 100 mmol/L CaCl₂.2H₂O for 30 min produced clear and uniform beads. Germination of encapsulated beads with shoot and roots was achieved on Murashige and Skoog (MS) medium augmented with 6-furfurylaminopurine (KN, 2.5 µmol/L) + α-naphthalene acetic acid (NAA, 1.0 µmol/L). For multiple shoot production, synseeds were incubated on 6-benzyladenine (BA, 5.0 µmol/L) + NAA (0.5 µmol/L) augmented MS medium followed by in vitro rooting on MS + indole-3-butyric acid (IBA, 1.0 µmol/L). The synseeds produced retained about 90% regeneration potential even after 4 weeks of storage at 4°C. Genetic stability of the regenerated plants was evaluated using 13 inter simple sequence repeats (ISSR) primers. The study thus provides an efficient system for production of synthetic seeds, their storage and subsequent conversion into genetically identical plants.     

Shoot tip culture of Ribes nigrum in vitro

A procedure of shoot tip culture for commercial production of plantlets of Ribes nigrum is described. An average multiplication rate of 4.7 proliferated shoots was achieved within 21 days of shoot tip (1-2 mm) cultures on a Murashige and Skoog (MS) medium supplemented with 1-2 mg 1“1 6-benzylaminopurine and 0.1 mg T1 indole-3-acetic acid. Following 1-3 d of dark treatment with three proliferated shoots per culture tube on half-strength MS medium, 83-96% of these shoots rooted. When these rooted shoots were transferred to wooden boxes with vermiculite as supporting medium for hardening, 97% survived. Plantlets grew well after transplanting to the nursery field. It is concluded that the use of (i) smaller shoot tip explants during shoot profileration stage, (ii) initial three days of dark treatment during the root initiation stage, and (iii) vermiculite as a supporting medium for plantlets during the hardening stage, are economic, efficient and practical procedures for commercial production of plantlets of R. nigrum by shoot tip cultures.     

Callus induction and plant regeneration from lateral shoots of herbaceous bamboo Mniochloa abersend

Callus induction and plant regeneration of Mniochloa abersend via lateral shoots were conducted in this study. Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L naphthaleneacetic acid (NAA) was effective for compact callus induction. Remarkably, calli on the MS medium with 0.1 mg/L 2,4-D yielded the highest folds of proliferation (8.01), and showed a high potential capacity to differentiate 1 year after subculture. In addition, the compact calli possessed 100% differentiation rate and generated more shoots that were green and strong in 1.0 mg/L kinetin and 1.0 mg/L NAA. Vigorous roots were generated in the 1/2MS supplemented with 0.5 mg/L indole-3-butyric acid, and the resultant plantlets exhibited 90% survival rate after they were hardened and transplanted. The established regeneration system of M. abersend provides a promising platform for bamboo gene function study.     

Meristem culture and micropropagation of a variety of ginger (Zingiber officinale Rosc.) with a high yield of oleoresin

Summary

Meristems of ginger with or without leaf primordia were induced to form shoots on three-quarter strength Murashige-Skoog’s (MS) medium containing sucrose 6%, coconut milk (CM) 20%, ascorbic acid (AA) 100 mg l^?1, glutamine (GL) 400 mg l^?1, activated charcoal (AC) 250 mg l^?1, 6-benzylaminopurine (BAP) 0.5 mg l^?1, indolebutyric acid (IBA) 0.4 mg l^?1 and agar 0.8%. Meristem-derived shoots exhibited consistent multiplication on three-quarter strength MS medium containing sucrose (3%), AA (100 mg l^?1), AC (100 mg l^?1), BAP (4–5 mg l^?1) and agar (0.8%). Liquid media (agitated or static) were less effective than a solid (agar-gelled) medium for micropropagation. Kinetin and naphthalene acetic acid (NAA) incorporated at various levels (0.01–0.8 mg l^?1) with or without added BAP and IBA neither improved plantlet formation nor enhanced shoot multiplication. The in vitro plants were successfully established in vivo and the rhizome yield was comparable with that of plants grown by conventional methods.     

In vitro regeneration and conservation of wild species of Arachis

Summary

Wild species of Arachis are restricted to South America and generally occur in regions under intensive environmental disturbance. Both in situ and ex situ conservation strategies are required in order to maintain the availability of these genotypes. This work developed in vitro regeneration systems from seed explants of 17 wild species of Arachis from six Sections (Heteranthae, Caulorrhizae, Triseminatae, Erectoides, Procumbentes and Arachis). After seed disinfection, embryonic axes, leaflets and cotyledons were excised aseptically and cultured on Murashige and Skoog (MS) medium supplemented with 8.8 µM, 22 µM or 110 µM 6-benzylaminopurine (BAP). Cultures were maintained in a growth chamber at 28° ± 2°C with a 16 h photoperiod. Regeneration patterns from seed explants were similar among species from all Sections. Embryonic axes produced plants through meristematic amplification or multiple shoot formation, while cotyledons and embryonic leaflets produced shoots at significantly lower frequencies through direct and indirect organogenesis, respectively. Shoots obtained from all explants were transferred to MS medium without growth regulators to induce root formation.     

A study of the effect of growth regulators and time of plantlet harvest on the in vitro multiplication rate of hardy and hybrid tea roses

The effect of benzyladenine (BA) (0, 0.5, 2.5, 5 and 10 ppm), naphthaleneacetic acid (NAA) (0,0.005,0.01,0.1 and 1 ppm) and time of proliferation (six, eight and ten weeks) on the optimum multiplication conditions for four Rosa hybrida cvs Champlain, John Franklin, John Paul II and Landora were studied. BA and NAA both had a significant effect on the proportion of viable plantlets and on multiplication rates while low or high concentrations of these growth regulators in the medium generally caused a reduction of plant material. The optimum multiplication rate also varied with cultivar and time. Concentrations of NAA above 0.1 ppm decreased the multiplication rate significantly for two of the cultivars. There was no consistent response between multiplication rates and cultivars to concentrations of B A, NAA or even the time of culture, and it is therefore recommended that each of these variates be determined for each cultivar to obtain optimum plantlet yield.