Effects of activated charcoal effects on induction and development of microtubers in potato (Solanurn tuberosum L.)

The aim of this research was to compare hormone-free medium with media with regulator substances (activated charcoal, cytokinins, polyamine biosynthesis inhibitor and chlorocholine chloride) used for microtuber induction and development. Explants of cvs Monalisa, Primura and Spunta were multiplied subculturing nodal segments on plant growth regulator-free Murashige & Skoog (1962) (MS) medium. When the plantlets had 6–8 nodes, single-node stem segments were excised and transferred to eight tuberisation media, each consisting of MS basal components supplemented with sucrose (8% w/v) and various regulator substances. The control was a regulator-free medium including only sucrose. Results were expressed as the number and weight of microtubers per nodal explant.
The cultivars showed wide variations in the mean weight of microtubers, ranging from 44.6 mg (Primura) to 77.5 mg (Spunta), and nearly all plants produced tubers. Medium containing activated charcoal gave the highest rate of tuberisation and the largest microtubers. It thus played a role in optimising conditions for rapid, mass tuberisation of these cultivars, and produced large microtubers for field planting.     

Relative importance of maltose and sucrose supplied during a 2-step potato microtuberization process

A 2-stage in vitro tuberization process comprising first micropropagation via nodal explants and then tuber induction in the resultant in vitro plantlets was studied using 2 cultivars of potato, Iwa and Daeji. In particular, the effects on both plantlet growth and subsequent in vitro tuberization of Murashige and Skoog (1962) basal medium containing either sucrose or maltose, each at 3 % (w/v), used for micropropagation were investigated. Sucrose and maltose were found to be equally effective in supporting development of vigorous plantlets from the nodal explants of both potato cultivars. Upon transfer to a medium with an optimised level of sucrose (i.e. 8 %, w/v) for in vitro tuberization, only the plantlets previously grown in the sucrose-containing medium were capable of forming more microtubers of the larger size category (greater than 0.5 g). The relative importance of sucrose supply at the mircropropagation stage was further confirmed when the resultant plantlets grown in the 3 % sucrose-containing medium were transferred to an in vitro tuberization medium containing either sucrose or maltose, each at 8 % (w/v). In this experiment, maltose and sucrose had indistingushable effects on in vitro tuberization.     

In vitro induction of minitubers in yam (<Emphasis Type="Italic">Dioscorea cayenensis</Emphasis>- <Emphasis Type="Italic">D. rotundata</Emphasis> complex)

Two methods were used to produce yam minitubers from two different yam cultivars (cv. Krengle and cv. Kponan) using in vitro culture techniques. Method 1: Yam microtubers were first initiated in vitro and then transplanted to soil to generate plants from which minitubers were produced. Yam plants were obtained either by directly planting the microtubers to soil, or by inducing the germination of the microtubers using various chemical and physical treatments, before their transfer to soil. Method 2: Yam plantlets were first produced in vitro and then transplanted to soil for further development and tuber production. In both methods, the presence of jasmonic acid (JA) in the culture medium was found to be essential for yam tuberization, as well as for the germination of yam microtubers. In vitro production of yam microtubers was variety dependant. Compared to cv. Krengle, cv. Kponan responded better to microtuberization, and 2.5 μM JA was the optimum concentration resulting in 70 and 90% explants producing microtubers in the MS medium and the Tuberization medium (T-medium), respectively. Germination of the microtubers required treatment of JA at concentrations ranging from 1.0 to 2.5 μM. The overall length of the process to produce minitubers from microtubers took 32 weeks. In contrast, minitubers were obtained within 20 weeks when plantlets were directly transferred to soil. In this case, plantlets were first grown for 8 weeks on medium containing JA (0.1–1.0 μM) and 8% sucrose to initiate plant growth and rooting.     

Effects of reducing sugar concentration on in vitro tuber formation and sprouting in yam (<Emphasis Type="Italic">Dioscorea cayenensis–D. rotundata</Emphasis> complex)

The effects of reducing sucrose level on tuber formation (% of cultures with microtubers), development (length and fresh weight of microtubers) and sprouting in yam Dioscorea cayenensis–D. rotundata complex in vitro were investigated. Only 29% of the explants showed tuber formation after 3 weeks in the presence of 1% sucrose in contrast to 100% with 3%. After 120 days of culture, the length and the weight of the tubers obtained in the presence of 1% sucrose were less than with 3% sucrose. Addition of sorbitol to keep osmolarity at the same level did not restore normal rate of tuber formation. Similar results were obtained with the use of reduced fructose or glucose level. Microtuber sprouting was also affected by sucrose level incorporated into the tuberisation medium. Tubers obtained on reduced sucrose level sprouted later and the increase of osmolarity with sorbitol did not restore normal sprouting. The bigger tubers obtained on high sucrose media could contain more carbohydrate reserves that could partially explain a higher sprouting rate. These results can be used for optimising in vitro conditions for mass production of microtubers in yam and especially in Dioscorea cayenensis–D. rotundata complex, a very important species in West Africa. They specially showed the importance of tuberisation conditions on precocity of tuberisation, on tuber length and weight and on their further sprouting.     

Optimisation of plantlet regeneration from leaf and nodal derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews

An indirect in vitro plant regeneration protocol for Vanilla planifolia has been established. Juvenile leaf and nodal segments from V. planifolia were used as explants to initiate callus. Nodal explants showed better callus initiation than juvenile leaf explants, with 35.0% of explants forming callus when cultured on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l 1-naphthylacetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA). Almost 10.0% of juvenile leaf explants were induced to form callus on the MS basal medium containing 2.0 mg/l NAA and 2.0 mg/l BA, whereas no callus formed in the presence of any concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and BA. After 8 weeks, callus generated was transferred to MS basal medium containing 1.0 mg/l BA and 0.5 mg/l NAA. A mean number of 4.2 shoots per callus was produced on this medium, with a mean length of 3.8 cm after 8 weeks of culture. Roots formed on 88.3% of plantlets when they were cultured on MS medium supplemented with 1.0 mg/l NAA, with a mean length of 4.4 cm after 4 weeks of culture. Of the rooted plantlets, 90.0% survived acclimatisation and were making new growth after 4 weeks.     

Nutrient alginate encapsulation of nodal segments of Althaea officinalis L., for short-term conservation and germplasm exchange

Abstract

In the present study, an alternate method for germplasm storage in the form of artificial seeds was standardized via nodal explants excised from in vitro proliferated shoots. The explants were encapsulated using sodium alginate and calcium chloride as gelling matrix. For development of root along with shoot, excised nodal segments were pretreated with ½ MS medium along with 20 μM IBA for 24 h and encapsulation was carried thereafter. Combination of 3% sodium alginate augmented with 100 mM CaCl₂.2H₂O was found appropriate for the formation of clear and uniform beads and subsequent conversion of encapsulated nodal segments into plantlets. Maximum (66%) encapsulated nodal segments were converted into plantlets on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA after eight weeks. Regeneration frequency of auxin-pretreated encapsulated and non-encapsulated nodal segments (stored at 4 ºC) was evaluated at different storage time (0 to 6 weeks). After four weeks of storage, encapsulated propagules exhibited highest conversion response on the optimized medium after eight weeks of culture. Plantlets were hardened and established with success in ex vitro conditions. Conversion of synthetic seeds into plantlets was observed when these were directly sown in autoclaved Soilrite^TM (Keltech Energies, Bangalore, India).     

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N ⁶-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.     

Somatic embryogenesis in clonal neem, <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss. and analysis for <Emphasis Type="Italic">in vitro</Emphasis> azadirachtin production

Summary Efficient and highly reproducible induction of somatic embryogenesis was obtained in four out of seven selected clones of neem, Azadirachta indica A. Juss. This was achieved either directly from root and nodal explants or indirectly from callus cultures initiated from leaf explants excised from 1-yr-old axenic plants. Direct induction of somatic embryogenesis was achieved both from nodal and root segments within 8 wk of culture on MS1 medium without growth regulators. However, the addition of 2.3–4.5 μM thidiazuron and 0.5 μM 2,4-dichlorophenoxyacetic acid into the medium were necessary to induce somatic embryogenesis via callus phase from leaf explants. Repetitive embryogenesis was observed within 3–4 wk following transfer of somatic embryos to a plant growth regulator-free medium. When somatic embryos of nodal and root segments were left on the induction medium without subculturing, approximately 15% of the somatic embryos developed into whole plantlets after passing through a series of developmental stages. Plantlets thus produced were hardy, lush green, and acclimatized casily under greenhouse conditions. However, somatic embryos derived from leaf explants showed low conversion rates (<5%). HPLC analysis revealed no detectable levels of azadirachtin in somatic embryos.     

In vitro regeneration of plantlets in Brassica alboglabra

Different vegetative parts of Brassica alboglabra seedlings and mature plants were used as explants in culture.A high frequency (60–100%) of shoot regeneration was obtained from hypocotyl explants, nodal stem segments, internodal segments and shoot apices cultured on Murashige-Skoog basal medium. Addition of 6-benzylaminopurine and kinetin increased the average number of shoots per explant. When detached and transferred to basal medium, the shoots readily developed roots. Regenerated plantlets could be successfully transplanted in soil.     

Jasmonate effects on in vitro tuberization and tuber bulking in two potato cultivars (Solanum tuberosum L.) under different media and photoperiod conditions

Summary Jasmonic acid (JA) effects on in vitro tuberization of potato nodal explants cvs. Sangre and Russet Burbank were tested under liquid and solid media conditions and 0,8, and 16h photoperiod. Explants taken from stock plants grown on 2.5μM JA-supplemented medium tuberized first, particularly in darkness. The most pronounced benefits of the JA pretreatment were recorded under 16h photoperiod, which is known to inhibit tuberization. Cultivar Sangre benefited from the JA preconditioning of stock plants more than Russet Burbank. Russet Burbank required the JA supplement in tuberization media to reach the same degree of stimulation. Overall, microtubers produced either from JA preconditioned stock plants or on the JA-containing tuberization media were more uniform and larger than from other treatments. Eight hours photoperiod was by far the best treatment for the production of high-quality uniform microtubers. JA conditioning of stock plants prior to taking explants for tuberization is being proposed as a treatment enhancing the quality of microtubers.     

Somatic embryogenesis and plantlet production using rejuvenated tissues from serial grafting of a mature <Emphasis Type="Italic">Kalopanax septemlobus</Emphasis> tree

An effective micropropagation technique via somatic embryogenesis has been developed using tissue from serially grafted shoots generated from a mature Kalopanax septemlobus tree (~40 y old). Callus was induced from leaf segments obtained from the grafts by culturing the explants in Murashige and Skoog (MS) medium supplemented with 2,4-D and 3% w/v sucrose under darkness. The effects of sucrose, coconut water, and polyethylene glycol (PEG-3350) were evaluated as factors to promote development of somatic embryos (SEs) from embryogenic callus. More than 90% of explants formed callus; however, only 2.5%, or 20 leaf segments out of 800 explants, formed embryogenic callus after 8 wk of culture. High sucrose concentrations (3% and 5% w/v) were effective in inducing SEs. Treatment with 2–10% v/v coconut water also had a positive effect on embryo induction. A synergistic effect on SE induction was obtained using sucrose and PEG, with presence of the latter compound resulting in smaller, more uniform SEs. Embryo germination and conversion to plantlets were significantly influenced by the gelling agents. In general, gelrite-gelled medium was superior to agar-gelled medium. In gelrite-gelled medium, gibberillic acid (GA₃) enhanced embryo germination. Converted plantlets in an artificial soil mixture showed a 91% survival rate and displayed no distinct morphological variations. Our results indicate that reliable somatic embryogenesis and plant production can be achieved with rejuvenated tissues after repeated grafting of shoots derived from a mature Kalopanax septemlobus tree.     

In vitro propagation and cytology of wild yams,Dioscorea abyssinica Hoch. and D. mangenotiana Miège

Two wild yams of West Africa, Dioscorea abyssinica Hoch, and D. mangenotiana Miège were micropropagated from nodal cultures. Both species produced 4–5 nodes per each node cultured. The size of nodal cuttings was critical, segments shorter than 0.5 cm being less suitable for micropropagation. The number of nodes produced was constant even after 5 cycles of subculture; however, D. abyssinica continuous subculture decreased propagation efficiency, resulting in a reduced number of reculturable nodes at each cycle. In D. mangenotiana, the decrease in multiplication efficiency affected both the number of total and reculturable nodes. Large-sized microtubers were induced on nodal segments maintained under 8-h daylength in both species. In D. abyssinica, however, microtubers were induced on media containing 20, 40, 60 and 80 g l^-1 sucrose, whereas in D. mangenotiana only 40 and 60 g l^-1 sucrose favoured tuberization. Cytological studies confirmed that the chromosome number of D. abyssinica was 2n=40, although a high incidence of cytochimerism and cells with 2n=38 were observed in root meristems. In D. mangenotiana clones, the chromosome number was 2n=40, as against 2n=72 and 2n=80 reported in literature. This species also displayed karyological stability.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - PPF photosynthetic photon flux     

Recurrent production of plants of black plum, Syzygium cuminii (L.) Skeels, a myrtaceous fruit tree, from in vitro cultured seedling explants

 The epicotyl segments bearing scaly leave(s), excised from in vitro grown seedlings of Syzygium cuminii, produced multiple shoots when cultivated on Murashige and Skoog's (MS, 1962) medium supplemented with different concentrations of BA (0–2 mg l^–1). The optimum response was recorded on the medium containing 1 mg l^–1 BA. An average of 8.6 shoots per explant were produced 60 days after inoculation, following transfer to fresh medium after 30 days. The shoots were excised, and the residual explants were transferred to fresh medium, where they again developed shoots. Up to five such passages resulted in the production of shoots from the repeatedly subcultured original explants. However, during the fifth passage, organogenic response was negligible and the explants turned brown thereafter. Following repeated harvesting of shoots and subculture of the residual explants, an average of 29 shoots per explant was obtained. The in vitro developed shoots produced roots when transferred to Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The developed plantlets were planted in soil and transferred to fields after an acclimatization period of 7–8 months. These plants have been thriving well for more than 3 years. The nodal explants excised from in vitro developed shoots and plants also exhibited a similar response when cultured on MS+1 mg l^–1 BA. Thus, a protocol has been developed to raise plants of S. cuminii at any time of the year. Received: 1 December 1998 / Revision received: 1 July 1999 · Accepted: 12 July 1999     

Carboxylic acids affect induction,development and quality of potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) microtubers grown <Emphasis Type="Italic">in vitro</Emphasis> from single-node explants

The role of three carboxylic acids with increasing alkyl-chain length, viz., formic, acetic and propionic acids in microtuberization was investigated in three potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of these carboxylic acids (0.0, 1.5, 3.0, 4.5 and 6.0 mM) were supplemented in microtuber induction medium, which was based on MS medium containing 8% sucrose, and their efficacy for induction, development and quality of microtubers was studied using single-node explants under continuous darkness at 20 °C. The carboxylic acids exhibited a strong stolon- and root-inhibiting effect on single-node explants with their increasing concentrations as well as alkyl-chain length (i.e., formic < acetic < propionic acids), and their mode of action was synonymous with antigibberellin substances. However, they did not have any significant inductive effect on microtuberization as compared to that under 8% sucrose medium. Rather they did show a detrimental effect on microtuber development in terms of average microtuber fresh weight with increasing concentrations as well as alkyl-chain length; both acetic and propionic acids at 6.0 mM induced the smallest microtubers in vitro. The carboxylic acids could, however, significantly increase the harvest indices suggesting their possible role in the regulation of source-sink co-ordination during microtuberization from single-node explants. But the most favourable effect of carboxylic acids on microtubers was apparent in terms of dry matter concomitant with higher starch synthesis and enhanced accumulation of reducing and total sugars. Acetic acid was the most effective in increasing the percentage of microtuber dry matter. The higher percentage of dry matter with higher carbohydrate reserves in microtubers induced by the carboxylic acids could be assumed to affect the quality of microtubers for subsequent storage, dormancy release and sprout growth.     

大花飞燕草的组织培养及再生体系建立

分别采用种子切段、无菌苗真叶作外植体首次成功建立了大花飞燕草的组织培养和再生体系。结果表明：大花飞燕草种子切段和无菌苗真叶大多数是通过愈伤组织途径再生,也有极少数不经过愈伤组织阶段而直接再生出小植株。在合适的培养基上,种子切段和叶片两种外植体离体培养均能高效再生,平均每个外植体能分化出１０－２０个不定芽。种子切段培养的最适通用培养基为改良ＭＳ附加ＺＴ３ｍｇＬ＾－１和ＮＡＡ ０．３ｍｇＬ＾－１,叶培     

In vitro flowering of indica rice (Oryza sativa L. spp. indica)

Nodal explants of rice cultivar Pathumthani 1 (PT1; short-day photoperiod insensitive) were collected, surface-disinfected, and cultured on modified MS medium under in vitro conditions for 90 d. A total of 60% nodal explants generated flowering plantlets (with one inflorescence per cluster). The net photosynthetic rate was greater, and soluble sugars (including glucose, fructose, and sucrose) accumulated to higher levels in the leaves of flowering as compared to non-flowering plants. In contrast, chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoid content were enriched to a greater degree in the leaves of non-flowering as compared to flowering plants. Also, growth performance parameters, including plant height, number of leaves per plant, leaf area, fresh weight, and dry weight of plantlets derived from seedlings were superior to those of plantlets derived from nodal explants. In addition, the protocol proved to successfully induce flowering in KDML 105, a short-day photoperiod-sensitive rice cultivar.     

Influence of preculture treatment and types of explants on shoot growth and in vitro flowering of feathered amaranth (Celosia argentea var. plumose)

The shoots developed from both the shoot tip and nodal explants of feathered amaranth (Celosia argentea var. plumosa—feathered cockscomb or plumed cockscomb) after 8 weeks of culture in the presence of either paclobutrazol or benzyladenine (BA) were shorter than those developed on basal Murashige and Skoog (MS) medium (Physiol Plant, 15:473–497, 1962) alone. However, this retarding effect was more pronounced in the nodal explant culture. Shoot tip explants from 2-week-old seedlings were more adversely affected by 0.85 or 1.7 μM paclobutrazol than those from older seedlings. In contrast, regardless of preculture duration investigated nodal explants did not exhibit different response to three different concentrations of paclobutazol. The response to 2.2 or 4.4 μM BA appeared to be largely independent of the age of the shoot tip explants or preculture treatment of nodal explants. Shoots developed from nodal explants produced a higher number of terminal inflorescence than those from shoot tip explants. Moreover, only lateral shoots from nodal explant culture formed inflorescence. Increased preculture duration on basal MS medium could generally lessen the inhibitory effect of lower concentrations of paclobutazol or BA on terminal or lateral inflorescence formation in nodal explant culture.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

Micropropagation of camphor tree (Cinnamomum camphora)

Multiple shoots were induced from shoot tips and nodal segments of a 12-year-old tree of Cinnamomum camphora on Woody Plant Medium (WPM) supplemented with BA and kinetin. The nodal segments from the in vitro developed plantlets could be induced again to produce a large number of harvestable shoots. Harvested shoots were rooted in vitro in WPM supplemented with activated charcoal (AC) and IBA. The plantlets were transferred to thermocol cups after which they were replanted into polybags and then to field. These plants survived with over 90% success under field conditions and exhibited vigorous growth. This system could be utilized for large-scale multiplication of C. camphora by tissue culture.