Production of adventitious shoots and plantlets from the hypocotyl explants of Sesbania rostrata (Bremek & obrem)

Summary Adventitious shoots were induced from the hypocotyl explants derived from 12–15-d-old seedlings of Sesbania rostrata on Nitsch's medium (Nitsch, 1969) supplemented with 1 mgl⁻¹ (4.4 μM), of N⁶-benzylademine (BA). A maximum of 5.9±3.4 shoots per explant in 100% of cultures were obtained. The BA treatment for different time durations (1, 3, 5, 7, 10, 17, 21, or 30 d) exhibited significant variation in the caulogenic potential of the explants. BA treatment for 10–17 d proved optimum for the response. Although at all concentrations of kinetin the explants developed multiple shoots, they were malformed. Sucrose at 3% exhibited the development of the maximum of 3.5±0.9 shoots per explant with an average shoot length of 4.7±3.9 cm. Among the different carbon sources, i.e., fructose, galactose, maltose, mannose, and sucrose at 3% (w/v), sucrose supported the best caulogenic response. The role of various other factors (viz. size, orientation of explant, and seedling age) on the caulogenic response of the hypocotyl explants of S. rostrata were also studied. The shoot development in all cases was accompanied by the development of moderate to profuse callus at the basal cut end of the explant. The in vitro-regenerated shoots produced roots when transferred to half-strength MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose and 1 mgl⁻¹ (4.9 μM) indole-3-butyric acid (IBA). The developed plantlets were transferred to the field after an initial acclimatization period of 3–4 mo. Such transferred plants produced flowers and fruits in the field and exhibited the development of prominent and organized stem nodules.     

Regulatory role of iron and EDTA in the shoot development from the epicotyl explants of Syzygium cuminii

Iron has proved to be an integral component of the culture medium supporting the caulogenic response of the epicotyl segments of S. cuminii. In the absence of both the iron and EDTA even the shoot buds failed to develop, while in the presence of either of these, though the shoot buds developed, their elongation was adversely affected. Among the three iron sources tested, ferrous sulphate proved to be the best, as the ferric chloride was not as effective as the former when used either alone or along with EDTA. Ferric citrate, on the other hand, when provided alone, elicited better response than that induced by ferrous sulphate alone. However, in combination with EDTA, the response declined significantly. The estimation of endogenous levels of iron in the explants further supported these results. The quantum of iron absorption was at a maximum during the first week of the culture and the explants, once deprived of iron during the first week, failed to catch up to the level of iron accumulated in the explants maintained continuously on complete medium, even after transfer to the complete medium. Likewise, the level of copper ions did not come up to comparable levels even if the explants were transferred to the complete medium after initial deprivation.     

Axillary bud development of passionfruit as affected by ethylene precursor and inhibitors

Summary The effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and two inhibitors, silver thiosulfate (STS) and aminoethoxyvinylglycine (AVG), were tested in yellow passionfruit (Passiflora edulis f. flaricarpa Degener) acillary cultured in vitro. The organogenic responses were assessed by the number of buds per explant, mean leaf area per explant, and shoot length. ACC-supplemented medium significantly inhibited all evaluated responses at both concentrations tested. When ethylene action and biosynthesis were inhibited, a significant increase in the number of developed buds and average leaf area was observed. Accumulated ethylene and its accumulation rate were significantly greater at 10 μM ACC, with a maximum production rate deteeted: at the 14th day and a decline at the 21st day. The results suggest beneficial effects of ethylene inhibitors on in vitro development of axillary buds and their reliability for use as an alternative approach to evaluate sensitivity of Passiflora species to ethylene. Even though shoot elongation did not differ from that of the control, the inhibition of the ethylene action and its biosynthesis by AVG and STS, respectively, significantly enhanced the number of buds per explant and leaf area.     

Direct organogenesis from leaf explants of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> and cultivation in bioreactor

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 μM of N ⁶-benzylaminopurine and kinetin ranging from 4.65 to 6.98 μM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm⁻³ sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 μM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm⁻³ sucrose resulted in a high biomass yield of 50.68 g dm⁻³ (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 μM, 15 g dm⁻³ sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.     

High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledons of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L.

The effect of copper sulphate on differentiation and elongation of shoot buds from cotyledonary explants of Capsicum annuum L. cv X-235 was investigated. Shoot buds were induced on medium supplemented with 22.2 μM BAP and 14.7 μM PAA. Elongation of shoot buds was obtained on MS medium containing 13.3 μM BAP + 0.58 μM GA₃. Both shoot induction and elongation media were supplemented with different levels of CuSO₄ (0–5 μM). The levels of CuSO₄ in the induction as well as elongation medium highly influenced the shoot bud formation and their subsequent elongation. Highest number of shoot buds per explant was obtained when the concentration of CuSO₄ was increased 30 times to the normal MS level. Shoot buds formation frequency i.e., the number of shoots formed per explant was increased two fold as compared to those formed on control. Elongation both in terms of percentage and length of shoots was better than that on control. Healthy elongated shoots were rooted on MS medium supplemented with 5.7 μM IAA. Rooted plantlets were transferred to field conditions.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

Somatic embryogenesis from the axillary meristems of peanut (Arachis hypogaea L.)

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with 90.5 μM 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with 13.6 μM 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.     

In vitro propagation of cashew from young trees

Summary Regeneration of cashew (Anacardium occidentale L.) from shoot explants of young grafts of mature tree origin is described. Establishment of shoot cultures was affected by season of collection, source, and type of explant. Explants from young grafts established better than those collected from field trees, and nodal cuttings regenerated better than shoot tips. Maximum percentage bud break and minimum contamination was noticed when shoots were collected in dry months (January to May). Pre-conditioning of stock plants by hormonal spray with 6-benzyladenine (BA) and gibberellic acid (GA₃) and brief presoaking of shoots in BA had no significant effect on culture establishment. MS (Murashige and Skoog, 1962) medium with half-strength major nutrients, 2.74 mM l-glutamine, 87.6 mM sucrose, and 2.25 gl⁻¹ phytagel was ideal for culture initiation. Inclusion of 0.1% polyvinylpyrrolidone (PVP-360) in the media reduced phenolic exudation. Solidified media was superior to liquid medium. Sucrose/glucose as energy source was found essential in the medium and had significant effect on percentage bud break and shoot development. A repeatable axillary shoot-bud induction was obtained on the above basal medium containing thidiazuron (TDZ) alone and in combination with BA. TDZ at 0.45 μM was best for axillary shoot-bud proliferation (4.5 buds per shoot) with maximum response (100%). Bud elongation could be stimulated in multiple shoots on medium containing 116.8 mM sucrose. In vitro rooting on auxin media and pulsing microshoots in 10 mM naphthalenaacetic acid (NAA) was ineffective. Rooting inability was, however, overcome by a micrografting procedure.     

In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

High frequency plant regeneration from thin cell layer explants of Brassica napus

Explants composed of the epidermis and 4–9 layers of subepidermal cells were excised from internodes of Brassica napus L. ssp. oleifera cv. Westar and cultured on modified Murashige and Skoog (MS) medium. The three or four terminal internodes excised from plants at an early stage (before any flower buds had opened) were shown to be the best explant source. Both cytokinin and auxin were required for induction of shoot organogenesis. Of six auxins tested, only naphthaleneacetic acid (NAA) was effective in shoot bud initiation. All four cytokinins tested (when associated with 0.5 mgl^-1 NAA) promoted organogenesis, but at differing frequencies. The highest shoot induction frequency was obtained at 10–15 mgl^-1 benzyladenine (BA). The organogenic response was strongly affected by the nitrogen content of the medium. The best response was observed when NO₃ ^- was the sole nitrogen source (supplied as KNO₃) in the range 30–90 mM. Sucrose and glucose were equally supportive in shoot regeneration with the optimal levels at 0.12 M and 0.15 M, respectively. Shoots were rooted on medium free of growth regulators and mature plants were grown in the greenhouse. Plants were also recovered from leafy structures which differed morphologically and histologically from shoot buds.     

Micropropagation and field evaluation of micropropagated plants of turmeric

A protocol was developed for in vitro propagation of turmeric cv `elite' using young vegetative buds from sprouting rhizomes. The shoot buds produced multiple shoots when cultured on MS solid medium supplemented with benzyladenine and 1-naphthalene acetic acid. The effect of various cytokinins on shoot multiplication was studied by culturing the shoot tips on MS liquid medium supplemented with benzyladenine, benzyladenine riboside, kinetin, kinetin riboside, zeatin, 6-,-dimethylallylaminopurine, adenine, adenine sulfate or metatopolin each at 10 M in combination with 1-naphthalene acetic acid (1 M). Significant differences were observed between the treatments. Liquid medium was more favourable than agar medium for shoot multiplication. Among the various concentrations of agar tested, 0.4% and 0.6% were the best and produced the highest number of shoots per explant. Among the different carbohydrates tested, sucrose, fructose, glucose, sugar cubes, maltose, levulose and market sugar were found to be equally effective for shoot multiplication and xylose, rhamnose, lactose and soluble starch were inhibitory. Ninety five percent of the micropropagated plants survived in sterilized soil in paper cups and all of them survived in the field. Among 48 plants, two plants showed variegated leaves on the tillers. The micropropagated plants showed a significant increase in shoot length, number of tillers, number and length of leaves, number of fingers and total fresh rhizome weight per plant when compared with conventionally propagated plants. RAPD analysis of 11 regenerated plants using sixteen 10-mer primers did not show any polymorphism.     

Factors influencing direct shoot regeneration from ovary explants of saffron

Direct adventitious shoot regeneration from ovary explants of Crocus sativus L. was influenced by media components, incubation conditions, and age of the explant. The best response towards caulogenesis (28%) with highest shoot numbers per ovary was observed when full strength Murashige and Skoog (1962) medium was supplemented with naphthaleneacetic acid and benzyladenine. Incubation in the dark at 20 °C was beneficial for induction of shoot buds. Ovaries of different growth stages having stigmas of pale yellow, pale orange and bright orange regenerated a maximum mean number (3.8 – 4.2) of shoots per ovary. Further development of ovary-derived shoots was influenced by the composition of basal salts in the culture medium where full strength Murashige and Skoog salts gave the best response of those tested. Regenerated shoots produced normal photosynthetic leaves and corms. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative studies of cytokinins on in vitro propagation of Bacopa monniera

A mass in vitro propagation system for Bacopa monniera (L.) Wettst. (Scrophulariaceae), a medicinally important plant, has been developed. A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants. Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (6.8 μM) and 6-benzyladenine (8.9 μM) proved superior to other treatments. Optimum adventitious shoot buds induction occurred at 6.8 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation. However, subculture of leaf explants on medium containing 2.2 μM benzyladenine yielded a higher number (129.1) of adventitious shoot buds by the end of third subculture. The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting. In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 4.9 μM IBA. After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rapid in vitro propagation of Aloe barbadensis Mill

Axillary bud development and adventitious bud formation was obtained with decapitated shoot explants of Aloe barbadensis Mill. Maximal bud growth and rooting of shoots was obtained on a modified medium of Murashige and Skoog supplemented with 5 M IBA. More adventitious and axillary buds developed on nutrient media supplemented with IBA than with NAA. Axillary buds but not adventitious buds developed with IAA in the medium. Morphogenesis was inhibited by 2,4-D. Kinetin, benzyladenine and thidiazuron were toxic to the explants and did not stimulate the development of axillary of adventitious buds. The optimal temperature for bud growth and development was 25°C. Axillary bud growth and the formation of adventitious buds was slowed down at 10°C and totally inhibited by 30°C. The optimal sucrose concentration was 3% with the inhibition of bud growth and development by higher sucrose levels.     

Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.)

Summary Studies were undertaken to optimize tissue culture conditions for micropropagation of Aleppo pine (Pinus halepensis Mill.) from mature embryos and various explants of the embryo. Over 90% of the embryo explants gave rise to adventitious buds within 4 wk. Intact embryos were the most suitable explants for shoot bud induction. Both isolated cotyledons and hypocotyls produced adventitious buds, but these developed slowly and failed to elongate. N⁶-Benzyladenine (BA) alone at 5.0μM was the most effective cytokinin when added to gelled to gelled von Arnold and Eriksson’s (AE) medium containing 3% sucrose. Adventitious bud development was achieved on hormone-free AE medium, and shoot elongation was optimum on three quarter-strength Bornman’s MCM medium, with 0.1% conifer-derived activated charcoal. Shoots were multiplied on three-quarter strength MCM medium, containing 5μM BA. To induce adventitious roots on the elongated shoots, pulse treatment with 1 mM IBA for 6 h, followed by the transfer of the shoots to sterile peat:vermiculite (1:1) mixture, was beneficial. After acclimatization for 3 to 4 wk under mist, almost all the rooted shoots could be transplanted successfully to the greenhouse, where the plants exhibited normal growth habit. Histologic studies on the ontogeny of adventitious shoot formation from mature embryo explants revealed temporal structural changes in different parts of the explant. Induction of mitotic divisions on the shoot-forming medium resulted in the formation of meristemoids in the epidermal and subepidermal layers of the explant, located initially at both the tips of the cotyledons and the axils of adjacent cotyledons. Shoot buds arising in the axils of adjacent cotyledons were due to new cell division and not to any preexisting meristem.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

Effect of phytoregulators and physiological characteristics of the explants on micropropagation of Maytenus ilicifolia

Micropropagated shoots of Maytenus ilicifolia Mart. were obtained from axillary buds cultured in Murashige & Skoog medium supplemented with 13.3 M 6-benzyladenine (BA). Addition of 1.1 M 1-indole-3-acetic acid (IAA) to the medium increased shoot elongation. The number of shoots formed was influenced by BA concentration, degree of juvenility of the explant, and by bud explant position on the stem. Cultures of buds taken from stem parts located close to the shoot tip yielded more callus than shoots, whereas axillary buds at distant positions from the apical bud yielded more shoots.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic-acid     

De novo organogenesis and plant regeneration in <Emphasis Type="Italic">Pongamia pinnata</Emphasis>, oil producing tree legume

Role of Thidiazuron (TDZ) in inducing adventitious organogenesis in Pongamia was studied. TDZ at different concentrations (0, 0.45, 2.27, 4.54, 6.71, 9.08, 11.35, 13.12 and 22.71 μM) were used for induction of caulogenic bud formation in deembryonated cotyledon explants. Each cotyledon was cut into three segments and identified as proximal, middle and distal. Duration of TDZ exposure, influence of the segment and orientation of the explant were studied. TDZ at 11.35 μM concentration was optimum for the induction of shoots and rapid elongation. Shoots induced at higher concentration elongated after several passages in growth regulator free medium, thereby extending the period of differentiation. Exposure of the explant for 20 days yielded more number of buds than 10 days. Proximal segment of the cotyledon was more responsive. Contact of abaxial surface in the medium was more effective and generated more buds than the adaxial side. Buds differentiated and elongated on transfer to MS basal medium for 8–12 passages of 15 days each. Rooting and elongation of shoots was achieved in charcoal supplemented half-strength MS medium. Rooted plantlets survived on transfer to sand soil mixture. The plants were hardened and transferred to green house. This is the first report on in vitro regeneration of Pongamia pinnata via adventitious organogenesis using TDZ. This protocol may find application in studies in genetic transformation, isolation of somaclonal variants and in induction of mutants. It also provides a system to study the inhibitory role of TDZ on shoot differentiation.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

In vitro corm production of Gladiolus dalenii and G. tristis

The ability of the summer flowering Gladiolus dalenii Van Geel and the winter flowering G. tristis L. to form corms in vitro was investigated. G. dalenii spontaneously formed corms on a shoot induction medium consisting of the basal medium of Murashige & Skoog (1962) with up to 2.0 mg l^-1 benzyladenine (BA), 3% sucrose and solidified with 2 g l^-1 Gelrite^®. The effect of different BA and sucrose concentrations as well as different temperatures on in vitro corm production of G. tristis was further investigated. The best production of shoots per explant was achieved on a medium containing 0.5 to 1.0 mg l^-1 BA, sucrose concentrations of 6 to 9% and cultured at 15°C. The best corm production was achieved at the same temperature and with the same medium with the exception that BA was omitted from the medium. To test the effect of the osmotic potential on the formation of shoots and corms, sucrose was substituted by mannitol at various concentrations. Sucrose proved to be essential for both shoot and corm production and the use of mannitol had no beneficial effect.