High Frequency Plantlet Regeneration Research Articles

Direct high frequency plantlet regeneration from leaf explants of Solanum torvum (swartz). A medicinally important plant

Direct high frequency plantlet regeneration from leaf explants of <i>Solanum torvum (</i>swartz). A medicinally important plant

Effects of plant growth regulators onin vitromicropropagation ofRuta chalapensisL.

Micropropagation is a rapid method for in vitro clonal multiplication of elite clones and also helps in dissemination and ex situ conservation of medicinal plants. In the present study, we report a highly efficient and cost effective protocol for high frequency plantlet regeneration from leaf explants of Ruta chalapensis L., commonly known as Fringed Rue which is an important medicinal herb. The leaf explants were cultured on Murashige and Skoog (MS) basal medium supplemented with Naphthalene acetic acid (NAA), 2,4-Dichlorophenoxyacetic acid (2,4-D), Indole-3-acetic acid (IAA) and Indole-3-butyric acid (IBA). MS medium supplemented with various concentrations of auxins such as IAA, IBA, NAA and 2,4-D was also tested. Among the various growth regulators used, NAA (1.0 mg/L) showed maximum callus induction (86.77±3.47%) from leaf explants, followed by NAA (2.0 mg/L) (79.34±3.09%). MS medium was fortified with NAA (0.5 mg/L) showed maximum (93.60±3.75%) of direct shoot induction. The concentration of auxins such as NAA increased by 2.0 mg/L produced lower shoot induction (74.10±2.96%). About 89.70±3.59% of roots were obtained when MS medium was amended with NAA (0.5 mg/L) as well as NAA (1.0 mg/L). Combination of NAA (1.5 mg/L) + KIN (2.0 mg/L) produced 77.02±3.08% of roots induction from leaf explants. Maximum green organogenic callus (95.55±3.82%) was observed in MS medium with NAA (0.5 mg/L) + KIN (0.5 mg/L). MS medium amended with IAA (0.5 mg/L) + KIN (0.5 mg/L) showed maximum (89.20±0.74%) of shoot induction from organogenic callus. MS medium amended with IAA (0.5 mg/L) + KIN (1.0 mg/L) showed maximum number (87.10±0.44%) of multiple shoot formation from organogenic callus. Maximum number (87.10±0.20%) of root formation from organogenic callus was obtained when the shoots were transferred to MS medium fortified with IAA (0.5 mg/L) + BAP (1.0 mg/L). The well-rooted plantlets were established on soil with 85% survival.

Shoot regeneration from leaf explants of Withania coagulans (Stocks) Dunal and genetic stability evaluation of regenerates with RAPD and ISSR markers

Withania coagulans (Stocks) Dunal (Solanaceae) is a critically endangered medicinal plant known for its multiple medicinal properties. We report here an efficient and high frequency plantlet regeneration system through direct organogenesis in W. coagulans using in vitro-derived leaves as explants. On Murashige and Skoog's (MS) medium containing 4.44μM 6-benzylaminopurine (BAP) 73.7±4.3% explants responded and produced 11.4±0.9 shoot buds per explant. The regenerated shoot buds were elongated (6.7±0.22cm) on MS medium with 1.11μM BAP and 0.57μM indole-3-acetic acid (IAA). The elongated shoots were rooted both in vitro and ex vitro. The regenerates were acclimatized by slow and gradual exposure to different regimes of temperature and relative humidity. The leaf-regenerates were assessed for genetic stability using RAPD and ISSR molecular markers and confirmed true-to-type. The in vitro regeneration system developed would be useful for genetic restoration program as the overexploitation and reproductive failure forced W. coagulans towards the verge of complete extinction. The regeneration method would also be useful for genetic transformation for genetic improvement and improved phyto-pharming.

High-frequency plantlet regeneration by somatic embryogenesis from mature zygotic embryos of onion

Somatic embryogenesis is a promising approach for plantlet regeneration. In order to establish a high-frequency plantlet regeneration system of onion in vitro, we used mature zygotic embryos as experimental material to improve the culture protocol. The results showed the highest induction rate of embryogenic callus (91%) was found when the NH4+/NO3– ratio in Murashige and Skoog (MS) medium was adjusted to 30/30 mM/mM. Well-proliferated embryogenic calli were observed in a 3 L airlift balloon-type bioreactor. During bioreactor culture, the continuous immersion culture was better. Inoculation density of 5 g/L and 0.1 vvm (air volume/culture medium volume per min) air volume were optimal for embryogenic callus proliferation. In addition, to obtain mature somatic embryos, the effects of sucrose and thiamine (VB1) were examined. Results showed that 20 g/L sucrose favoured somatic embryo maturation. When the VB1 concentration in normal MS medium (0.1 mg/L) was increased to 10 mg/L, the rate of somatic embryo germination reached 93.3%. When the mature somatic embryos were inoculated in MS medium without plant growth regulators, whole plantlets regenerated after 15 d of culture. During plantlet acclimatisation, the maximum survival rate (93.8%) was found in the substrate of 1/2 vermiculite and 1/2 perlite.

Direct somatic embryogenesis with high frequency plantlet regeneration and successive cormlet production in saffron (Crocus sativus L.)

Saffron is a slowly propagating geophyte with fungal infestation of corms under field conditions. Moreover, genetic improvement through breeding is not possible due to male sterility. Therefore, tissue culture methods offer a great potential for mass propagation, and somatic embryogenesis is one of the efficient methods. In the present study, direct somatic embryogenesis from leaf base was obtained in thidiazuron (TDZ, 2.5μM) and picloram (2.0μM) supplemented Murashige and Skoog medium (Murashige and Skoog, 1962). Significantly higher secondary embryogenesis was observed in embryo proliferation medium (EPM; MS supplemented with TDZ 2.5μM and picloram 1.0μM). The origin and different developmental stages of somatic embryos were ascertained through histological and scanning electron microscopic studies. Stomata were also observed in some of the somatic embryos. An alluring observation was retention of embryogenic potential beyond 3years of culture. Percent germination of secondary embryos in MS medium was 60.48 when matured in ½ EPM. Freshly initiated somatic embryos also germinated to form plantlets without undergoing secondary embryogenesis in MS medium containing TDZ (2.5μM) and picloram (1.0 and 2.0μM). Somatic embryo derived shoots were multiplied in 6-benzylaminopurine (BAP; 26.64μM) and α-naphthaleneacetic acid (NAA; 1.0μM). Growth performance of cormlets obtained from these shoots was evaluated under green house conditions. Somatic embryogenesis holds tremendous importance for mass propagation of saffron with formation of ultimate propagating material i.e., cormlets.

High frequency plantlet regeneration from nodal segment culture of female Momordica dioica (Roxb.)

An in vitro propagation method for female plants of Momordica dioica (Roxb.) has been established. The nodal segments were harvested and the cut ends of the explants were sealed with wax and then surface sterilized and cultured. Bud breaking occurred on Murashige and Skoog’s (MS) agar-gelled medium + 2.0 mg L−1 6-Benzylaminopurine (BAP) + 0.1 mg L−1 Indole-3 acetic acid (IAA). The cultures were amplified by passages on MS medium supplemented with 1.0 mg L−1 BAP + 0.1 mg L−1 IAA. Further, shoot amplification (29.2 shoots per vessel) was achieved by subculturing of in vitro regenerated shoot clump on MS medium + 0.5 mg L−1 BAP + 0.1 mg L−1 IAA. The micropropagated shoots were subsequently transferred for root formation on half-strength MS medium + 2.0 mg L−1 Indole-3 butyric acid (IBA) with 89% success rate. The in vitro-regenerated shoots were also rooted ex vitro with 34% success. These plantlets were hardened in the greenhouse and transferred to the field. The established protocol is suitable for true to type cloning of mature female plant of M. dioica.

Plantlet Regeneration from Callus Cultures of Selected Genotype of Aloe vera L.—An Ancient Plant for Modern Herbal Industries

Aloe vera L., a member of Liliaceae, is a medicinal plant and has a number of curative properties. We describe here the development of tissue culture method for high-frequency plantlet regeneration from inflorescence axis-derived callus cultures of sweet aloe genotype. Competent callus cultures were established on 0.8% agar-gelled Murashige and Skoog's (MS) basal medium supplemented with 6.0 mg l⁻¹ of 2,4-dichlorophenoxyacetic acid (2,4-D) and 100.0 mg l⁻¹ of activated charcoal and additives (100 mg l⁻¹ of ascorbic acid, 50.0 mg l⁻¹ each of citric acid and polyvinylpyrrolidone, and 25.0 mg l⁻¹ each of L-arginine and adenine sulfate). The callus cultures were cultured on MS medium containing 1.5 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kinetin (Kin), and additives with 4% carbohydrate source for multiplication and long-term maintenance of regenerative callus cultures. Callus cultures organized, differentiated, and produced globular embryogenic structures on MS medium with 1.0 mg l⁻¹ of 2,4-D, 0.25 mg l⁻¹ of Kin, and additives (50.0 mg l⁻¹ of ascorbic acid and 25.0 mg l⁻¹ each of citric acid, L-arginine, and adenine sulfate). These globular structures subsequently produced shoot buds and then complete plantlets on MS medium containing 1.0 mg l⁻¹ of 6-benzylaminopurine and additives. A hundred percent regenerated plantlets were hardened in the greenhouse and stored under an agro-net house/nursery. The regeneration system defined could be a useful tool not only for mass-scale propagation of selected genotype of A. vera, but also for genetic improvement of plant species through genetic transformation.

Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in Zoysia japonica

In this study, we have demonstrated that Zoysia japonica callus induced from mature seeds can produce high frequencies of plant regeneration and somatic embryogenesis, even following a prolonged period of subculturing. Initial callus cultures were induced from mature seeds of Japanese lawngrass (Z. japonica Steud.) incubated on a medium containing major N6 medium salts, minor Murashige and Skoog (MS) medium salts, and modified MS medium organic elements supplemented with 3 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01–0.02 mg L−1 6-benzyladenine. Compact callus were selected and subcultured monthly on a medium containing 2 mg L−1 2,4-D, 0.5 mg L−1 kinetin, 500 mg L−1 casein hydrolysate, 500 mg L−1 proline, and 500 mg L−1 myoinositol. Callus maintained in vitro for 18 mo could be induced to regenerate plantlets with a frequency of >90%. By contrast, 36-mo-old callus cultures failed to produce normal shoot regeneration. However, the addition of CuSO4 to the subculture media maintained >90% regeneration frequencies in such long-term callus cultures. Histological observations revealed that plant regeneration occurred both through somatic embryogenesis and organogenesis pathways. The ability to sustainable regeneration in long-term callus cultures will be valuable to the program of genetic transformation and somaclonal variant selection.

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L−1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.

High frequency plantlet regeneration from callus and artificial seed production of rock plant Pogonatherum paniceum (Lam.) Hack. (Poaceae)

Pogonatherum paniceum (Lam.) Hack. is a rock plant with good potential for vegetative recovery on naked lands. A high frequency in vitro regeneration system was developed for P. paniceum. Calli were induced from explants of mature seeds, seedlings, young leaves, and stem segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg L −1 2,4-dichlorophenoxyacetic acid (2,4-D), 2.0 mg L −1 α-naphthalene acetic acid (NAA) and 0.2 mg L −1 6-benzylaminopurine (BAP). High induction rates (59.57%) and regeneration rates (100%) were obtained from mature seed explants; calli were sub-cultured for over 2 years and still retained a high regenerative capacity. One seed explant resulted in 69,997 plants in 1 year. Shoot buds derived from calli were used for encapsulation in liquid MS medium containing 3% sucrose and two different alginate matrices (3% sodium alginate (w/v) + MS medium containing 3% sucrose and 3% sodium alginate + 1% activated carbon (w/v) + MS medium containing 3% sucrose) with a 20-min exposure to 2% CaCl 2 and 0.3% bavistin (w/v). The capsule with 3.0% sodium alginate (w/v) and 1% activated carbon (w/v) showed a higher conversion rate (61.58%) and stronger plantlets under non-aseptic conditions. These systems are useful for the rapid clonal propagation and dissemination of artificial seed material of P. paniceum for eco-recovery.

High frequency plantlet regeneration from cotyledonary node cultures of Aegle marmelos (L.) Corr.

High frequency plantlet regeneration was achieved in cotyledonary nodes of Aegle marmelos. Cotyledonary nodes from 1 mo. old in vitro grown seedlings of A. marmelos were cultured on Murashige and Skoog (MS) medium supplemented with benzyl adenine (BA) (0–8.8 μM), kinetin (KIN) (0–9.4 μM), and indole-3-acetic acid (IAA) (0–1.14 μM) either alone or in combinations. The highest regenerative response was observed on medium containing 6.6 μM BA + 1.14 μM IAA where approximately 86.6% of the cultures responded with an average shoot numbers of 487.5 per explant in 7-wk time. Cultures maintained on KIN-supplemented medium showed very poor response. In vitro responded shoots were transferred to root induction medium consisting of half-strength MS supplemented with auxins IAA, indole-3-butyric acid (IBA), or α-naphthalene acetic acid (NAA). Rooting was best in medium supplemented with 14.7 μM IBA. Rooted plantlets were acclimatized and transferred to the field with 80% survival rate.

High-frequency plant regeneration through cyclic secondary somatic embryogenesis in black pepper (Piper nigrum L.)

A high-frequency plantlet regeneration protocol was developed for black pepper (Piper nigrum L.) through cyclic secondary somatic embryogenesis. Secondary embryos formed from the radicular end of the primary somatic embryos which were originally derived from micropylar tissues of germinating seeds on growth regulator-free SH medium in the absence of light. The process of secondary embryogenesis continued in a cyclic manner from the root pole of newly formed embryos resulting in clumps of somatic embryos. Strength of the medium and sucrose concentration influenced the process of secondary embryogenesis and fresh weight of somatic embryo clumps. Full-strength SH medium supplemented with 1.5% sucrose produced significantly higher fresh weight and numbers of secondary somatic embryos while 3.0 and 4.5% sucrose in the medium favored further development of proliferated embryos into plantlets. Ontogeny of secondary embryos was established by histological analysis. Secondary embryogenic potential was influenced by the developmental stage of the explanted somatic embryo and stages up to "torpedo" were more suitable. A single-flask system was standardized for proliferation, maturation, germination and conversion of secondary somatic embryos in suspension cultures. The system of cyclic secondary somatic embryogenesis in black pepper described here represents a permanent source of embryogenic material that can be used for genetic manipulations of this crop species.

Plant regeneration from ryegrass ovules cultivated on endosperm-derived feeder cells

An efficient method for the regeneration of zygote-derived plants via ovule culture is desirable for overcoming postzygotic cross incompatibility as well as for the development of certain methods for genetic manipulation. High-frequency plantlet regeneration from ovules of Italian ryegrass (Lolium multiflorum Lam.) and a hybrid Italian/perennial ryegrass excised 1 to 4 days post pollination was obtained by culture on endosperm-derived feeder cells. Ovules excised 3 or 4 days after anthesis and grown on feeder cells generally regenerated about twice as frequently as ovules grown directly on nutrient medium. In one of the genotypes tested, ovules excised 1, 2 and 3 days post pollination developed into plantlets at percentages of 38.1, 52.0 and 52.8, respectively, using the feeder-cell system.

Plant Regeneraton from Callus Cultures of Switchgrass

Regeneration from cells or tissues cultured in vitro is a fundamental requirement for most applications of plant biotechnology. The objective of this study was to develop protocols in which plants could be regenerated with ease and in high numbers from tissue cultures of switchgrass (Panicum virgatum L.). Mature caryopses and young leaf segments of the cultivar Alamo were explanted onto agar solidified Murashige and Skoog medium containing 2,4‐dichlorophenoxyacetic acid (2,4‐D) and 6‐benzylaminopurine (BAP). BAP at 45 μM for mature caryopses and at 5 μA/for leaf explants in combination with 22.5 μM 2,4‐D produced high frequency plantlet regeneration by both organogenesis and somatic embryogenesis. The mode of regeneration was documented by light and scanning electron microscopy. One thousand regenerated plants have been established in the field for observation and evaluation.

High frequency plant regeneration in Heliconia psittacorum L.f.

High frequency plantlet regeneration was induced from callus derived from shoot tips and thin cross sections of in vitro cultures of Heliconia psittacorum L.f. Fine nodular callus was initiated in the dark on semisolid Murashige and Skoog medium containing 0.5 g/l activated charcoal, 1.0 g/l casein hydrolysate and 80 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Reducing the concentration of 2,4-D in the medium to 40 μM and subculturing at 6-week intervals enabled long-term maintenance of this regenerative callus. There was no appreciable loss in regeneration potential for over 18 months in these cultures. Development and regeneration of protocorm-like bodies into plantlets occurred when 2,4-D was withdrawn from the medium. Regenerated plants were successfully transferred to field conditions.

Comparaison des aptitudes morphogénétiques des étamines fertiles ou stériles d'Actinidia chinensis cultivées in vitro

High frequency plantlet regeneration is obtained by means of in vitro culture of neoformed roots arising from Actinidia chinensis Pl. stamens. The favourable influence of zeatin upon bud initiation from roots is demonstrated. Potential differences between stamens coming from ♀ or ♂ plants are also shown. On the same medium, sterile stamens exhibit a greater rooting potential than fertile ones, but the latter have a more pronounced callusing efficiency. These differences are transmittable through dedifferentiation; neoformed roots of ♂ origin have a weak bud regeneration ability, whereas roots of ♀ origin give rise to callus and buds. The implications of such observations in regard to the endogenous contents of auxins and cytokinins and their relations with the ♀ or ♂ nature of the plants are discussed. The histological origin of the different kinds of regeneration are also examined and point to the preservation of rooting potential by the bundle sheath of the stamens and of budding potential by the pericycle of neoformed roots. These data are considered in relation to the recent literature.