Proliferation of meristematic clusters in disposable presterilized plastic bioreactors for the large-scale micropropagation of plants

Summary Proliferation of meristematic clusters of several plants in an inexpensive airlift bioreactor system, consisting of a disposable presterilized light transmittable plastic film vessel is described. The optimal shape, size, and structural function of the disposable plastic bioreactor are based on the bubble column and airlift glass bioreactors. The disposable bioreactors are designed in a conical configuration with a single inoculation and harvest port and multiple use dispensing and mixing accessories. Shearing damage and foaming problems known to exist in bioreactors due to the plant's rigid cell wall and size were greatly reduced in the disposable plastic bioreactors. The disposable bioreactors were used for propagule proliferation and growth, using meristem and bud clusters of potato, fern, banana, and gladiolus. The clusters' biomass increased five-to eightfold over a period of 26–30 d, depending on the species. The clusters were separated mechanically by a chopper made of a grid of knives. The chopped propagules were inoculated to agar medium for further growth and developed into transplantable plants. In the case of gladiolus and potato, corms and tubers developed in a sucrose-elevated storage organ induction medium, respectively, after the initial formation of small shoots. The plantlets and storage organs were transplanted to an acclimation greenhouse and continued to grow with a 95–100% survival, depending on the species. Plant development was followed for a period of 16 wk in fern and 12–14 wk in potato, banana, and gladiolus and normal shoot and leaf growth was observed. The feasibility of large-scale liquid cultures for plant micropropagation is discussed.     

SIVB 2003 Congress Symposium Proceeding: Plant Growth and Sugar Utilization in an Agitated,Thin-Film Liquid System for Micropropagation

Summary Agitated layers of liquid medium were created on platform shakers in jars with 25–30 ml of medium (similar to conventional agar culture) rotating at 90 rpm. Thin films were scaled up in larger rectangular vessels on tilted shelves that periodically rock. In jars of liquid medium with a density of 180 explants per liter, multiplication rates of Hota tokudama var. ‘Newberry Gold’ were optimal with a media sucrose concentration of 5% [both with and without 1 μM benzyladenine (BA)]. Endogenous levels of soluble sugars were directly related to the concentration of sucrose in the medium. Three Hosta cultivars (‘Striptease’, ‘Minuteman’, and ‘Stiletto’) with plant densities of 40–200 explants per liter of medium were tested in larger, agitated, thin-film vessels in media with 5% sucrose and directly compared to agar medium. Higher rates of multiplication were observed in liquid than agar with the magnitude of the difference dependent on explant density. Pooled results for the three varieties with 200 explants per liter showed multiplication rates of 1.7x and 2.3x for agar and thin-film liquid, respectively. At 40 explants per liter, the multiplication rate was increased to 2.1x for agar and 3.4x for thin-film liquid. Sugar uptake was greater in liquid than agar and was greater in the higher densities, with the magnitude of the effect dependent on plant variety. Increased vessel size in the liquid, thin-film system and greater sugar uptake allowed more, larger plants to be harvested. Alocasia macrorrhizos was cultured in growth medium containing 1μM BA and 5% sucrose with plant densities in the range of 33–330 explants per liter. Dry weight and multiplication rate were greater in the liquid system than agar with the magnitude of the difference dependent on plant density. With approximately 165 explants per liter, and greater at the initiation of culture, plant density limited growth in both agar and liquid thin-film systems. In a multiplication medium (3 μM BA and 3 μM ancymidol) plant size was reduced by 50% and 60% (fresh weight) in liquid and agar, respectively. Initial density in the range of 165–330 explants per liter did not limit growth with the smaller plants in liquid or semisolid multiplication medium. Sugar uptake was greater in liquid than agar. While ample sugar was present in media for growth at any density on agar, sugar depletion was limiting growth at highest densities with the larger plants in liquid growth medium. In semisolid agar medium, sugar uptake by plants was more rapid than diffusion across the agar medium, resulting in non-equilibrium conditions following the culture cycle. In agitated, liquid medium, a greater transfer of sugars to plant tissue was related to accelerated growth.     

Compact callus cluster suspension cultures of Catharanthus roseus with enhanced indole alkaloid biosynthesis

Summary Compact callus clusters showing a certain level of cellular or tissue differentiation were established from Catharanthus roseus stem and leaf explants in a modified MS liquid induction medium supplemented with 5.37 μM α-naphthaleneacetic acid and 4.65 μM kinetin. In the induction medium most leaf explants developed into friable half-closed hollow callus clusters, whereas in the same medium containing 2,4-dichlorophenoxyacetic acid instead of α-naphthaleneacetic acid, most leaf explants were induced to form dispersed cell suspension cultures. Characteristics of these different types of suspension cultures were compared, and the results showed that the compact callus clusters could synthesize indole alkaloids 1.9- and 2.4-fold higher than the half-closed hollow callus clusters and dispersed cell cultures, respectively. The degree of compaction expressed by the ratio of fresh weight to dry weight of these suspension cultures was correlated to indole alkaloid production. Our studies also postulated that the level of cellular/tissue differentiation might be responsible for these different alkaloid synthesis capabilities. Sucrose regime affected some properties (the size, degree of compaction, differentiation level) of the compact callus cluster cultures and therefore influenced alkaloid production.     

Abscisic acid and ancymidol promote conversion of somatic embryos to plantlets and secondary embryogenesis inAsparagus officinalis L.

Summary The effects of abscisic acid (ABA) (0, 0.09 μM, 0.19 μM, 0.28 μM, and 0.38 μM) or ancymidol (0, 0.98 μM, 1.95 μM, 2.93 μM, 3.90 μM) in embryo germination medium on the conversion of primary embryos to plantlets and secondary embryogenesis were evaluated for asparagus. ABA and ancymidol each significantly enhanced both responses. ABA was more effective than ancymidol in promoting the conversion of primary embryos to plantlets, while the converse was true for the production of secondary embryos. The most effective treatments for embryo conversion were 0.19 and 0.28 μM ABA; 75–77% bipolar and 55–57% globular embryos converted to plantlets. For secondary embryogenesis, the most effective treatments were 1.95 and 2.93 μM ancymidol; 99–101 and 84–86 somatic embryos were produced from 10 globular and 10 bipolar embryos, respectively. Bipolar embryos generally converted to plantlets better than globular embryos, but more secondary embryos were produced from globular embryos than from bipolar embryos in all treatments. ABA and ancymidol also affected the morphology of the plantlets produced. The plantlets from the embryos incubated on the medium with ancymidol had strong and thick shoots and roots, while those on the medium with ABA had long, thin shoots and short thin roots.     

Optimization of nutrient levels in the medium increases the efficiency of callus induction and plant regeneration in recalcitrant indian barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) <Emphasis Type="Italic">in vitro</Emphasis>

Summary The present study reports that a revised nutrient concentration in the basal medium improved shoot bud induction and subsequent plant regeneration in barley (Hordeum vulgare L. var. BL-2). Cultures were raised from immature embryos on MSB₅ medium supplemented with picloram. Concentrations of five nutrients were varied. The effect of these nutrients was investigated on (1) induction, (2) induction and subculture, and (3) induction, subculture and regeneration stages. The basal MSB₅ medium was not optimal for each phase of barley culture. Decreased ammonium nitrate, increased potassium dihydrogen phosphate, sodium molybdate, cobalt chloride, and addition of glycine enhanced shoot bud induction and plant regeneration. The different media that were optimal for immature embryo culture were: MSB₅ medium supplemented with 20.70 μM picloram, 10.30 mM NH₄NO₃, 6.25 mM KH₂PO₄, 2.06 μM Na₂MoO₄, 0.55 μM CoCl₂, and 26.64 μM glycine (for induction); MSB₅ medium supplemented with 12.47 μM picloram, 10.30 mM NH₄NO₃, and 0.55 μM CoCl₂ (for subculture); and MSB₅ medium supplemented with 0.2 μM picloram and 10.3 mM NH₄NO₃ (for regeneration). Primary cultures required 6wk (without transfer) for morphogenic callus formation. Callus required 4wk of subculture and another 4wk on regeneration medium for optimal plant regeneration. The revised medium could also promote regeneration of the recalcitrant barley genotype RD-2552. Histological analysis showed that the major pathway of differentiation was through shoot bud formation.     

Efficient somatic embryogenesis and plant regeneration from long-term cell suspension cultures of Medicago Truncatula cv. Jemalong

Summary Plants were suecessfully régenerated via somatic embryos from 3-yr-old cell suspension cultures of Medicago truncatula Gaertin. cv. Jemalong line M9-10a. The cultures were originally initiated from callus induced in well-expanded leaflets of 30 d in vitro-grown plants, Suspension cultures were established in stirred-liquid Murashige and Skoog (MS) basal salts and vitamins supplemented with 2.3 μM 2.4-dichlorophenoxyacetic acid (2,4-D) and 2.3 μM kinetin (Kin) and subeultured weekly. Somatic embryogenesis induction step was conducted in liquid MS medium containing 0.45 μM 2,4-D and 0.91 μM zeatin (Zea), during 1,2, and 3wk after subculture. Induced and non-induced cultures were transferred to solid embryo proliferation medium [EPM-MS basal salts and vitamins solidified with 0.2% (w/v) gelrite]. Somatic embryos developed until the late torpedo/dicotyledonary stages. We found that the best condition for the development of somatic embryos was achieved when suspension cultures were not subjected to the induction step. Induction of 1 and 2 wk led to a decrease in the recovery of somatic embryos and the 3-wk treatment resulted in no differentiation of somatic embryos. Plant regeneration was obtained in all conditions (except for 3wk induction) when embryos were transferred to an embryo conversion medium [ECM, similar to EPM but solidified with 0.7% (w/v) agar]. Embryo conversion rates were 54.5±1.6, 52.5±18.5, and 41.6±8.4% for 0, 1, and 2 wk induction treatments, respectively. These plants were successfully transferred to the greenhouse where they matured and produced seeds.     

Silver nitrate promotes shoot development and plant regeneration of chile pepper (Capsicum annuum L.) via organogenesis

Summary Chile pepper (Capsicum annuum L.) plants were regenerated from cotyledon explantsin vitro in four major stages: bud induction, bud enlargement, shoot elongation, and root development. Bud induction medium contained 0.5 mg/L (2.9μM) indole-3-acetic acid and 2 mg/L (8.9 μM) N⁶-benzyladenine. Bud enlargement occurred, and an occasional shoot appeared when medium with 2 mg/L (6μM) gibberellic acid, 2 mg/L (8.9 μM) N⁶-benzyladenine, and 5 mg/L (29.4 μM) silver nitrate was used. Most shoots elongated after placement on a third medium without plant growth regulators or on fresh plates of bud enlargement medium. Incubations were for 2, 2, and 4 weeks, respectively, at 28.5°C and continuous light. Treatment with silver nitrate was necessary for multiple shoot production and elongation to occur in the third culture stage and was most effective when present in the second-stage medium but not in the bud induction medium. Sixteen to 26% of the shoots rooted in medium with 1 mg/L (5.4 μM) 1-naphthaleneacetic acid after 1 month. Additional shoots transferred to a second rooting medium with 0.1 or 1.0 mg/L (0.54 or 5.4 μM) 1-naphthaleneacetic acid developed roots, increasing the overall rooting efficiency to 70–72%. Most rooted shoots grew well and produced viable seeds when grown in the greenhouse. Other cytokinins tested for plant regeneration were zeatin and thidiazuron. Zeatin induced few shoots and fewer well-developed plants. Thidiazuron induced multiple shoots 4 months after culture began, but many were small and did not elongate further. Phytagar tissue culture grade proved superior to other agars tested, increasing bud induction frequency from 0-33% to 80–93% and eliminating explant hyperhydricity.     

adventitious shoot regeneration from petiole explants of Heracleum candicans wall

Summary A method for adventitious shoot induction from petiole explants of Heracleum candicans is reported. Shoot buds were induced on Murashige and Skoog (MS) medium with 4.4μM 6-benzylaminopurine (BA) and 1.1 μM 2,4-dichlorophen-oxyacetic acid (2,4-D). A wound response in the presence of BA and 2,4-D at the time of culture was necessary for inducing shoot buds. The shoot bud regeneration was significantly influenced by size, type and orientation of explants on the culture medium. These shoot buds developed into 4–5 cm shoots upon transfer to a medium containing 1.1μM BA and 0.5 μM α-naphthaleneacetic acid (NAA). The regenerated shoots formed rooted plantlets on MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 15 plants were established in the field for further evaluation.     

Bud proliferation and plant regeneration in liquid-cultured philodendron treated with ancymidol and paclobutrazol

Philodendron plants propagated in liquid shake or bioreactor cultures proliferated profusely in the presence of paclobutrazol (PAC) and to a lesser extent in the presence of ancymidol (ANC). The growth retardants inhibited leaf development and induced the formation of bud clusters. Short transient treatments with low concentrations (1.7–3.4 M) of the growth retardants limited leaf growth and proliferation to a lesser extent than higher concentrations (6.8–17 M). The growth retardants had a carryover dwarfing effect in the semi-solid hardening medium, which was more pronounced at the higher concentrations or prolonged exposure periods. Regenerated plants resumed normal growth 3–6 weeks after transplanting. Treatment with growth retardants may become a useful method in the prevention of abnormal leaf growth in large-scale liquid cultures, as well as in enhancing bud proliferation.     

Plant regeneration protocol of medicinally important <Emphasis Type="Italic">Andrographis paniculata</Emphasis> (Burm. F.) Wallich <Emphasis Type="Italic">ex</Emphasis> Nees via somatic embryogenesis

Summary In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-1) on induction, maturation, and conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction, efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05μM 2,4-D started to form embryos at 135, 105, 150, and 185d, respectively, after explant establishment. Callus initiated at 13.56μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced concentration of 2,4-D (2.26μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2.4-D. The addition of 11.7μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar inedium with 0.44 μMN⁶-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant.     

Mint essential oil can induce or inhibit potato sprouting by differential alteration of apical meristem

Sprouting of potatoes during storage, due to tuber dormancy release, is associated with weight loss and softening. Sprout-preventing chemicals, such as chlorpropham (CIPC), can negatively impact the environment and human health. Monthly thermal fogging with mint (Mentha spicata L.) essential oil (MEO) inhibited sprouting in eight potato cultivars during large-volume 6-month storage: the tubers remained firm with 38% lower weight loss after 140 days of storage. The sprout-inhibitory action may be nullified: treated tubers washed with water resumed sprouting within days, with reduced apical dominance. MEO application caused local necrosis of the bud meristem, and a few weeks later, axillary bud (AX) growth was induced in the same sprouting eye. MEO components analysis showed that 73% of its content is the monoterpene R-carvone. Tubers treated with synthetic R-carvone in equivalent dose, 4.5 μl l⁻¹, showed an inhibitory effect similar to that of MEO. Surprisingly, 0.5 μl l⁻¹ of MEO or synthetic R-carvone catalyzed AX sprouting in the tuber. To the best of our knowledge, this is the first report of an essential oil vapor inducing early sprouting of potato tubers. R-carvone caused visible damage to the meristem membrane at sprout-inhibiting, but not sprout-inducing doses, suggesting different underlying mechanisms. After 5 days’ exposure to R-carvone, its derivatives transcarveol and neo-dihydrocarveol were found in buds of tubers treated with the inhibitory dose, suggesting biodegradation. These experiments demonstrate the potential of MEO vapor as an environmentally friendly alternative to CIPC in stored potatoes and as a research tool for the control of sprouting in plants.     

Overcoming phenolic accumulation during callus induction and in vitro organogenesis in water chestnut (Trapa japonica Flerov)

Summary Procedures for callus induction and subsequent organogenesis in the aquatic plant, water chestnut (Trapa japonica Flerov), were established. Phenolics exuded from explants at the callus-induction stage adversely affect callus growth. For cotyledonary node-derived callus cultured in Murashige and Skoog (MS) medium (full, half or quarter strength) containing 2,4-dichlorophenoxyacetic acid (2,4-D) alone or in combination with benzyladenine (BA), the accumulation of phenolics was reduced and callus induction increased by the addition of 10.8 μM phloroglucinol (PG) to the medium. Ascorbic acid was also effective in reducing phenolic accumulation, but less effective for callus induction than PG. Half-strength MS medium supplemented with 2.7 μM 2,4-D, 108.0 μM casein hydrolyzate, and 10.8 μM PG supported maximum callus induction. Plant organogenesis was increased by addition of vitamins (0.27 μM biotin and 2.7 μM folic acid) to half-strength MS medium supplemented with 0.27 μM BA. Many shoots developed from the regenerated nodal shoot explants in liquid half-strength MS salts medium supplemented with 1.08 μM BA and 0.27 μM naphthaleneacetic acid. Individual shoots were excised and cultured in liquid half-strength MS medium supplemented with 5.4 μM IBA and rooted plantlets (108) were transferred and acclimatized in plastic pots. After 3 wk, the plantlets were transplanted in a water chestnut field and the survival rate was 100%.     

Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media

 The regeneration potential of excised aspen (Populus tremula L.) roots cultivated in liquid medium, as affected by plant growth regulators and by the position of the isolated root explant on the main root, was investigated. The effect of various levels of benzyladenine (BA) and thidiazuron (TDZ) on bud regeneration in root explants was studied. TDZ in the medium had a marked effect on bud development as compared with BA, inducing a tenfold increase in the number of buds regenerated from various root explants. TDZ enhanced both root and root-borne shoot biomass production but reduced further shoot development and elongation. The position of the isolated root sections on the main root affected regeneration, the proximal sections further away from the root tip producing the highest number of buds per explant in both BA and TDZ treatments. Buds regenerated in close proximity to the site of lateral roots in BA-treated roots, while in TDZ-treated root sections, the buds formed all over the root regardless of the presence of lateral roots. The buds developed from inner cortical and sub-epidermal cell layers, disrupting the epidermis and the inner layers. Root biomass production and growth was greatly enhanced in well-aerated bioreactor culture in the presence of 4.5×10^–2 μM TDZ. A high number of the root-borne shoots could be rooted and converted to plantlets. However, while shoots regenerated in a medium with BA rooted well in a growth regulator-free medium, shoots formed in a medium with TDZ required auxin for rooting. Roots cultured in the presence of ancymidol, a gibberellin biosynthesis inhibitor, regenerated non-hyperhydric bud clusters and hyperhydric shoots. These were separated mechanically, subcultured to growth and rooting medium and transplanted ex vitro resulting in phenotypically true-to-type plantlets. The potential of liquid cultures for aspen shoot biomass production from roots is discussed. Received: 24 January 2000 / Revision received: 6 March 2000 / Accepted: 7 March 2000     

Micropropagation ofHesperaloe parviflora

Summary We successfully micropropagatedHesperaloe parviflora from mature plants. Shoot cultures were directly initiated from mature plants using pedicel bud explants on a modified Murashige and Skoog medium containing Nitsch and Nitsch vitamins and 1 μM zeatin riboside. Axillary shoot multiplication from established cultures was most responsive to changing concentrations of N⁶-benzyladenine (BA) with the greatest production of 6 μM BA. Growing shoots on a medium supplemented with 6 μM BA for 6 wk and then transferring cultures to a 1 μM BA medium for 6 more wk increased the number of transferable shoots, but not significantly. However, our data predicts that the maximum number of transferable shoots produced from a single microshoot would occur on media with 5.4 μM zeatin riboside. Shoots rooted easilyin vitro orex vitro and rooted shoots were easily acclimatized. The methods described in this paper are being used to commercially micropropagateH. parviflora.     

In vitro somatic embryogenesis and plant regeneration in Zantedeschia hybrids

A method for regeneration of plants from tuber explants of a Zantedeschia hybrid via somatic embryogenesis was developed. In vitro cultures were initiated starting from both anthers and tubers. Somatic embryogenesis was only achieved from tuber explants. 6-Benzyladenine (BA) at 0.6 or 2 mg l⁻¹ in combination with 2 mg l⁻¹ α-naphthaleneacetic acid (NAA) yielded the highest number of embryos per explant. The somatic embryos converted into plantlets on Murashige and Skoog basal medium supplemented with vitamins, micro- and macronutrients, 1 mg l⁻¹ 6-τ-τ-(dimethylallylamino)-purine (2iP), 3% sucrose and 0.7% agar. This is the first report on induction of somatic embryogenesis in Zantedeschia.     

High-frequency plant regeneration from cotyledon callus of <Emphasis Type="Italic">Acacia sinuata</Emphasis> (Lour.) Merr

Summary A new reliable protocol for the induction of adventitious shoots and plant regenertion from cotyledon-derived callus of Acacia sinuata has been developed. Calluses were induced from cotyledon explants on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar or 0.15% phytagel, 8.1 μM α-naphthaleneacetic acid, and 2.2 μM 6-benzylaminopurine (BA). High-frequency regeneration of adventitious buds from callus was achieved when cultured on half-strength MS medium supplemented with 10% coconut water, 13.3 μM BA, and 2.5 μM zeatin. Histological studies revealed that the regenerated shoots originated from the callus. Among the various carbohydrates tested, sucrose at 87.6 mM was optimum for shoot-bud induction. Addition of 1.7 μM gibberellic acid along with 4.4 μM favored shoot elongation. In vitro-raised shoots produced prominent roots when transferred to half-strength MS medium supplemented with 7.4 μM indole-3-butyric acid. Rooted plants, thus developed, were hardened and successfully established in soil (45%). This protocol yielded an average of 40 plantlets per cotyledon explant over a period of 3 mo.     

Plant regeneration from cotyledonary explants of jute, Corchorus capsularis L.

A liquid culture protocol was developed to regenerate shoots from cotyledons of germinating seeds of jute (Corchorus capsularis L.). Reproducibility of the protocol was tested in three genotypes of jute. Highest bud differentiation rates into normal shoots (via shoot-forming explants) were obtained on modified Murashige and Skoog's liquid medium containing 2.7 μM α-naphthaleneacetic acid and 4.4 μM benzylaminopurine. Regenerated shoots were excised, and the best root formation could be induced in medium with 2.5 μM indole-3-butyric acid and 1.5% sucrose. Bud primordia were formed directly on the cut surface of the cotyledons. Scanning electron micrographs and histological studies confirmed the organogenic nature of the regenerated shoots. The physical condition of the culture medium and the age of the explants played crucial roles in the induction of shoot development using shoots; 2-day-old explants being optimal. Approximately 70% of the shoots were successfully established in soil after hardening. Received: 20 October 1997 / Revision received: 4 October 1998 / Accepted: 27 October 1998     

Direct plantlet regeneration from male inflorescences of medicinal yam (Dioscorea floribunda Mart. & Gal.)

Summary Segments of male inflorescences of medicinal yam (Dioscorea floribunda) cultured on Murashige and Skoog (MS) medium supplemented with 13.94 μM kinetin (Kn) resulted in the conversion of floral buds into vegetative buds and these later developed into plantlets. Growth and multiplication of shoots could be obtained by culturing individual shoots in MS modified basal medium, replacing the MS standard three vitamins with 10.0 mgl⁻¹ thiamine in addition to 13.94 μM Kn. Root induction was also obtained simultaneously from the base of the shoots in the same medium. Such plantlets have been successfully transferred to potted soil, where they grew normally. Plantlets were also made to develop tubers on MS medium with 18.91 μM abscisic acid (ABA) and also with 2.68 μM α-naphthaleneacetic acid (NAA) and 40–50 gl⁻¹ sucrose.     

Plant regeneration through somatic embryogenesis in medicinally important <Emphasis Type="Italic">Centella asiatica</Emphasis> L.

Summary High-frequency somatic embryogenesis and plant regeneration was achieved on callus derived from leaf (petiole and lamina) and internode explants of Centella asiatica L. Growth regulators significantly influenced the frequency of somatic embryogenesis and plant regeneration. Calluses developed on Murashige and Skoog (MS) medium fortified with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 5.37 μM α-naphthaleneacetic acid (NAA), both with 2.32 μM kinetin (Kn), were superior for somatic embryogenesis. Callus developed on NAA and Kn-supplemented medium favored induction and maturation of embryos earlier compared to that on 2,4-D and Kn. Embryogenic callus transferred from NAA and Kn-supplemented medium to suspension cultures of half-strength MS medium with NAA (2.69 μM) and Kn (1.16 μM) developed a mean of 204.3 somatic embryos per 100 mg of callus. Embryogenic callus transferred from 2,4-D and Kn subsequently to suspension cultures of half-strength MS medium with 2,4-D (0.45 μM) and Kn (1.16 μM) developed a mean of 303.1 embryos per 100 mg of callus. Eighty-eight percent of the embryos underwent maturation and conversion to plantlets upon transfer to half-strength MS semisolid medium having 0.054 μM NAA with either 0.044 μM BA or 0.046 μM Kn. Embryo-derived plantlets established in field conditions displayed morphological characters identical to those of the parent plant.     

Plant regeneration via organogenesis from adventitious bud explants of a medicinal herb species, <Emphasis Type="Italic">Polygonatum cyrtonema</Emphasis>

Summary Explants derived from adventitious buds, rhizomes, stems, and leaves of a medicinal plant, Polygonatum cyrtonema, were studied for plantlet regeneration, and only adventitious bud explants were able to be regenerated into plantlets. Regeneration was also accompanied by the formation of rhizome-like tissue, the medicinal portion of the plant. The optimum hormone combination for plantlet regenertion was 4.44 μM benzyladenine plus 2.26 μM 2,4-dichlorophenoxyacetic acid, at which new adventitious buds were obtained from 96.6% of the adventitious bud explants, with an average of 5.2 buds per explant. The best medium for root induction was half-strength Murashige and Skoog medium with 4.57 μM α-naphthaleneacetic acid, as 92% of regenerated buds rooted. Regenerated plantlets were successfully transferred to a greenhouse with 86% survival. Histological observation indicated that new adventitious buds originated from the superficial meristematic cell cluster of the granular callus induced from adventitious bud explants via organogenesis.