Multiple shoot regeneration in seed-derived immature leaflet explants of peanut (Arachis hypogaea L.)

A protocol was developed for organogenesis from immature leaflet explants derived from mature seeds of peanut. Immature leaflets pre-incubated on MS medium supplemented with 13.32 μM BAP + 4.95 μM NAA for 7 days, turned green and enlarged. The enlarged green leaflets produced multiple shoot buds after 1–2 cycles of sub-culture on MS medium supplemented with 13.32 μM BAP. Three cycles of shoot buds on the elongation medium (13.32 μM BAP) produced 6.17 ± 0.47 elongated shoots per explant. The shoot bud formation was genotype independent. All elongated shoots rooted on the medium containing 4.95 μM NAA. The complete protocol gave efficient (>81%) direct organogenesis, leading to the development of plantlets within 4 months.     

A micropropagation system for cloning of Bambusa tulda Roxb.

The communication describes standardization of an efficient in vitro propagation and hardening procedure for obtaining plantlets from field grown culms of Bambusa tulda. Administration for 10 min of 0.05 and 0.1% mercuric chloride to explants collected in winter and summer seasons, respectively facilitated optimum culture establishment and bud break. 0.1–0.2% mercuric chloride in rainy season enhanced aseptic culture establishment but inhibited bud break due to toxicity to explants. MS liquid medium enriched with 100 μM glutamine, 0.1 μM indole-3-acetic acid and 12 μM 6-benzylaminopurine supported maximum in vitro shoot multiplication rate of two-fold. The proliferated shoots were successfully rooted on MS liquid medium supplemented with 40 μM coumarin resulting in a maximum of 98% rooting. The procedure requires 45 days cycle for the in vitro clonal propagation (15 days for shoot multiplication and 30 days for root induction) and 80 days for acclimatized plantlet production.     

In vitro regeneration from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit

Methods to regenerate whole plants from mature leaf explants of Pelargonium rapaceum (L.) L’Hérit were established. To optimize shoot induction, leaf explants were cultured on media containing different types and combinations of plant growth regulators. Growth was initiated within 17–24 days culture, and included callus formation, and root or shoot organogenesis ranging from 20 to 100% regeneration. Shoots were induced only when explants were cultivated on MS medium containing a combination of NAA and kinetin, NAA and BAP, IAA and Kinetin, or IAA and BAP. On media containing NAA and BAP, dark incubation was critical for efficient direct shoot regeneration from explants. Direct shoot formation and the highest number of shoots per explant (17.6) were obtained from leaf explants cultured in the dark for 30 days on MS medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ BAP. Shoots cultured on MS medium containing 0.1 mg l⁻¹ NAA formed tuberous roots with microtubers within 42 days. Healthy regenerated plants were acclimated and transferred to a greenhouse.     

Micropropagation of gooseberry, Ribes grossularia

A method for rapid micropropagation of gooseberry (Ribes grossularia cultivar ‘Hinnonmäki Yellow’) was established. The optimum concentration for shoot production was obtained at 2.2 μM BAP and 0.05 μM IBA with MS nutrient medium. The Lepoivre salt medium with no hormones was used for elongation of shoot clusters, and in this medium about 50% of the shoots formed roots. For both rooted and unrooted shoots, 100% survival was obtained after transfer to soil. It was possible to store shoots in vitro for 130 days without any sub-culture and with 100% survival if the temperature was increased successively after cold storage.     

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% CaCl₂ 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.     

Commercial production of Cordyline terminalis (L) Kunth. from shoot apex meristem and assessment for genetic stability of somaclones by isozyme markers

Rapid multiplication of Cordyline terminalis (L) Kunth. was achieved from shoot apex explant on Murashige and Skoog's (MS) medium supplemented with 3% (w/v) sucrose and different concentration of growth regulators at various combinations. On MS medium supplemented with BA in combination with Adenine sulphate and IAA, shoot initiation and multiplication were obtained. Best elongations of shoots were found on 1/2 MS basal medium and shoots were rooted on 1/2 MS medium supplemented with IBA. Rooted plants passed through a hardening phase prior to ex vitro transfer. Clonal propagated plants established in garden soil were uniform and identical to mother plant in respect to growth characteristics and morphology. Isozymic profiles of different micropropagated clones were assessed for their genetic stability. Ten clones were tested for six isozymes. Only a few showed variation with respect to the banding pattern in esterase and superoxide dismutase. In superoxide dismutase, the two polymorphic isoforms (Rf 0.06 and 0.45) appeared in the clone C8 of the plants transferred to the field after 15 subcultural passages. Mobility and intensity of bands were monitored in other isozymes. Isozyme markers may be used as a tool for rapid screening of genetic stability in tissue cultured clones of C. terminalis.     

Effect of 2,4-d, glutamine and BAP on embryogenic suspension culture of date palm (Phoenix dactylifera L.)

The proliferation of embryogenic suspension culture in two cultivars (Jihel and Bousthami Noir) of Phoenix dactylifera L. was tested on liquid media with or without 2,4-d and with different glutamine concentrations (3.35 × 10⁻⁴, 6.7 × 10⁻⁴ and 13.4 × 10⁻⁴ M). The liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine has clearly improved the proliferation of somatic embryos. In fact, when glutamine concentration increased from 3.35 × 10⁻⁴ to 6.7 × 10⁻⁴ M, the yield of somatic embryos increased from 14 to 56 embryos per 100 ml of culture medium for “Jihel” cultivar and 25–71 embryos per 100 ml of culture medium for “Bousthami Noir” cultivar. In contrast, increasing glutamine concentration from 6.7 × 10⁻⁴ to 13.4 × 10⁻⁴ M, the embryos yield was negligible. Based on biochemical analysis, the highest accumulation of proteins and sugars was obtained in liquid medium with 0.1 mg l⁻¹ 2,4-d and 6.7 × 10⁻⁴ M glutamine (118 and 91 mg of proteins g⁻¹ DW, respectively, for “Jihel” and “Bousthami Noir” cultivars; 194 mg of sugars g⁻¹ DW for “Jihel” cultivar and 182 mg of sugars g⁻¹ DW for “Bousthami Noir” cultivar). In addition, the supply of 0.05 mg l⁻¹ BAP on the germination medium could be useful in terms of germination percentage of somatic embryos. When BAP concentration increased from 0.05 to 0.2 mg l⁻¹, the germination percentage of somatic embryos decreased from 14.2 to 4.9%, while secondary embryogenesis increased from 26.4 to 45.2%.     

Effect of gibberellic acid (GA3) on elongation and rooting of ‘St. Julien A’ rootstock in vitro

‘St. Julien A’ (Prunus instititia L.) rootstock was induced to proliferate shoots on a modified half-strength Murashige and Skoog (MS) medium. Cultures treated with 12.5 mg l^?1 gibberellic acid (GA₃) produced elongated shoots suitable for rooting. Elongated shoots were placed in media with indolebutyric acid (IBA) or indole-3-acetic acid (IAA) with or without a 16-day dark incubation. Light (16-h photoperiod) inhibited rooting. IAA (4 mg l^?1) was ineffective in promoting rooting. Rooting was best when shoots were incubated in the dark with IBA (4 mg l^?1). GA₃ was deleterious to shoots, causing chlorosis and apical die-back. Light regime interacted with auxin treatments in affecting shoot condition. Shoot condition was better on shoots treated with IBA and dark-incubated; while those treated with IAA were better when light-incubated.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

Significance of explant preparation and sizing in Aloe vera L.—A highly efficient method for in vitro multiple shoot induction

The present study was carried out to assess the effect of explant preparation and sizing for in vitro micropropagation of Aloe vera L. The stem nodal explants and shoot tips were cultured on modified Murashige and Skoog's medium (1962) supplemented with different concentrations of 6-benzylaminopurine (BA), kinetin (KIN), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) either singly or in combination. The best media composition was found to be MS medium supplemented with IAA (11.42 μM), IBA (9.8 μM) and BA (8.88 μM). The explants were divided into 2 sets, with and without ensheathing leaf base. Explant sizing, pruning and retention of mother tissue was highly significant in induction of multiple shoots and roots. The stem nodal explants with leaf base performed much better than those without such covering. A very high number of shoots and roots grew from these explants. The rooted plantlets were successfully acclimatized and transferred to the green house conditions and finally to field conditions.     

Plant regeneration of pansy (Viola wittrockiana) ‘Caidie’ via petiole-derived callus

An in vitro plant regeneration protocol for pansy (Viola wittrockiana) cultivar ‘Caidie’ from petioles was established as following: callus induction on a half-strength MS medium supplemented with 0.45 μmol l⁻¹ 2,4-d plus 8.9 μmol l⁻¹ BA, callus subculture on medium F (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 0.44 μmol l⁻¹ BA) and then on medium T (1/2MS with 4.5 μmol l⁻¹ 2,4-d, 2.7 μmol l⁻¹ NAA and 2.2 μmol l⁻¹ BA), shoot regeneration on medium D3 (MS media supplemented with 2.9 μmol l⁻¹GA₃, 23.6 μmol l⁻¹ AgNO₃, 0.02% active charcoal and 4.5 μmol l⁻¹ TDZ), shoot multiplication on medium M (half-strength MS medium containing NAA 1.1 μmol l⁻¹, TDZ 9.1 μmol l⁻¹ and GA₃ 8.7 μmol l⁻¹), and then shoot elongation and rooting on medium R (MS medium supplemented with 1.1 μmol l⁻¹ NAA and 1.1 μmol l⁻¹ BA). Subculture on appropriate medium was found to be important for successful shoot regeneration.     

Rapid regeneration of plants of Dendrobium lituiflorum Lindl. (Orchidaceae) by using banana extract

Effects of banana extract (BE) and 6-benzylaminopurine (BAP) were evaluated on asymbiotic seed germination and an early differentiation of protocorms and plant regeneration of Dendrobium lituiflorum Lindl. High percentage germination was achieved by culturing seeds on modified Knudson C medium supplemented with 10% (v/v) BE. Rapid regeneration was observed within 60 days of culture on 10% (v/v) BE supplemented KC medium where maximum percentage propagules showed development of leaves and root formation. Propagules on BAP supplemented KC medium showed no further development beyond one leaf stage. In another experiment, culture of shoots on 12.5% (v/v) BE supplemented KC medium led to multiplication, shoot elongation as well as vigorous rooting. Shoots cultured on 10 μM BAP supplemented MS medium showed maximum multiplication but these were stunted. Plants with well expanded deep green leaves and elongated roots from BE media were first hardened in vitro followed by ex vitro hardening on cocopeat:perlite (9:1) in the greenhouse conditions and exhibited 90% survival. The study emphasizes the role of BE as a natural additive at different stages of development from seed germination to plant regeneration.     

Regeneration from leaf explants and protoplasts of Brassica oleracea var. botrytis (cauliflower)

Adventitious shoot regeneration and protoplast isolation and culture were examined from leaf explants of in vitro shoot cultures of several cauliflower (Brassica oleracea var. botrytis) cultivars, sourced from Europe and Australia, was investigated with the aim to develop improved nuclear and plastid transformation protocols for this vegetable crop. Eight out of 10 cultivars regenerated shoots from at least 79% of leaf explants. Mesophyll protoplasts from leaves gave high yields and division frequencies. Growth of shoot cultures in large glass vessels with vented lids was the key factor in obtaining high protoplast division frequencies of up to 71% and at least 70% of protoplast calluses regenerating shoots.     

In vitro propagation of Lychnis senno Siebold et Zucc., a rare plant with potential ornamental value

Lychnis senno is a rare and valued ornamental plant. Seed propagation is not efficient because of the low germination rate. To grow commercially L. senno in China, a protocol for in vitro germination and propagation of this species was developed. Various germination rates were obtained by treating seeds with GA₃ during 1–6 months storage period. The highest germination rate reached 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4 °C. Axillary shoot proliferation was induced in the nodal segments of the seedlings on medium containing specific concentrations of BA and NAA [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497]. Maximum number of shoots was developed on a medium supplemented with 5 mg/l BA and 0.5 mg/l NAA, while the higher shoots were observed on a medium supplemented with 0.5 mg/l BA and 0.05 mg/l NAA. Rooting was induced in 91.7% of the regenerated explants on a half-strength MS medium supplemented with 0.5 mg/l NAA. The plantlets grew well and flowered after transfer to the greenhouse. The chromosome numbers of seedlings and propagated plants were also determined to be 2n = 2x = 24.     

Regeneration and characterization of Swertia chirata Buch.-Ham. ex Wall. plants from immature seed cultures

First generation immature seeds (R₁) were collected from a field transferred micropropagated plant and seeds were induced to develop organogenic calli in Swertia chirata, a traditional revenue earning medicinal plant. Half strength MS medium with different growth regulators namely, BA, Kn (2.22–4.44 μM), NAA (2.69–5.37 μM), and 2.26 μM 2,4-D were used to induce callus and organogenesis. Isolated shoots produced roots either in the same medium or in presence of NAA (2.69–10.74 μM) or IBA (2.46–9.8 μM). Fully developed plantlets were successfully transplanted to soil and the fertile seed bearing plants developed. Occasionally plants derived from more than 56 weeks old calli showed some morphological variations. Such variations in regenerated plants is not reflected in their chromosomal constitution, with normal 2n = 26 chromosomes. Likewise, no variation was observed in DNA fingerprinting patterns among the short-term raised culture regenerants, which were morphologically similar to that of the donor plant illustrating their genetical uniformity and clonal fidelity. On the contrary, variation in DNA fingerprinting patterns was observed in long-term culture raised plants.