High frequency shoot regeneration from cotyledon explants of cucumber via organogenesis

Organogenic callus induction and high frequency shoot regeneration were achieved from cotyledon explants of cucumber. About 86.2% of cotyledon explants derived from 5-day-old in vitro raised seedlings produced green, compact nodular organogenic callus in MS medium containing NAA (2.69 μM) and BA (4.44 μM) after two successive transfers at 20 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MS medium supplemented with NAA (1.34 μM), BA (8.88 μM), zeatin (0.91 μM) and l-glutamine (136.85 μM) with shoot induction frequency of 75.6%. Shoot proliferation occurred when callus with emerging shoots was transferred in the same medium at an interval of 20 days. Shoots (1.0 cm length) were excised from callus and were elongated in MS medium fortified with GA₃ (1.44 μM) and BA (4.44 μM). The elongated shoots were rooted in MS medium supplemented with IBA (3.42 μM) and BA (4.44 μM). Rooted plants were acclimatized in green-house and subsequently established in soil with a survival rate of 80%. This protocol yielded an average of 35 shoots per cotyledon explant in a culture duration of 120–140 days.     

Micropropagation of Capsicum frutescens L. using axillary shoot explants

A novel micropropagation protocol was established for Capsicum frutescens L. cv. ‘Uchithi’, a pungent chilli cultivar, through induction of axillary shoot proliferation of in vitro raised plantlets by decapitation and using the axillary shoots as explants for multiple shoot bud induction. About 2–6 axillary shoots were induced within 2 weeks when 4-week-old in vitro raised plantlets were decapitated. The axillary shoot-tip explants produced multiple shoot buds when cultured on Murashige and Skoog's (MS) medium containing 8.8–44.4 μM 6-benzylaminopurine (BAP) or 9.3–46.7 μM kinetin alone or 8.8–44.4 μM BAP with 4.6 μM kinetin or 5.7 and 28.5 μM indole-3-acetic acid (IAA). Maximum number of shoots (5.6) were induced on medium containing 22.2 μM BAP in combination with 4.65 μM kinetin. The separated shoots rooted and elongated on medium containing 2.8 μM IAA or 2.4–4.9 μM indole-3-butyric acid (IBA). Rooted plantlets were successfully established in the soil. Efficient mass multiplication of this important food crop was achieved.     

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.     

Restoration of rooting competence in a mature plant of Garcinia indica through serial shoot tip grafting in vitro

Apical and axillary buds from a high yielding, early fruiting elite tree (more than 20 years old) were cultured in woody plant medium (WPM) supplemented with 0.9 μM N⁶-benzyl adenine (BA). Multiple shoots were obtained on WPM basal medium containing 8.9 μM BA and 0.5 μM thidiazuron (TDZ). Elongation of axillary shoots was obtained in half-strength WPM medium supplemented with 0.4 μM BA. For root initiation, the elongated shoots were transferred to half strength WPM basal medium containing 2.5–245 μM indole-3-butyric acid (IBA) or 2.7–268.5 μM α-naphthaleneacetic acid (NAA) or the shoots were subjected to 2.5–53.9 mM IBA, 2.7–59.1 mM NAA dip for (30 s–30 min) and then transferred to half strength WPM basal medium. However, rooting was never achieved even after 2 months of culture.     

Direct and callus-mediated regeneration of Curcuma soloensis Valeton (Zingiberaceae) and ex vitro performance of regenerated plants

Protocols are outlined for the regeneration of Curcuma soloensis, an attractive tropical ornamental plant, from young vegetative bud explants. We used both direct and callus-mediated regeneration techniques to produce material suitable for mass propagation and the development of transgenic plants. During direct plantlet propagation, the presence of thidiazuron (TDZ) in the growing medium induced more than three times as many shoots as 6-benzylaminopurine (BA), with a mean of 18.7 shoots per explant on MS medium containing 2.5 μM TDZ compared to 5.0 shoots with 40 μM BA. Subsequently, the shoots rooted readily on MS basal medium that was free of plant growth regulators. During indirect plantlet regeneration, TDZ combined with BA and 2,4-dichlorophenoxyacetic acid (2,4-D) had significant effects on embryogenic callus induction and multiplication. The frequency of callus formation was 91.1% for explants cultured on MS basal medium supplemented with 2.5 μM TDZ, 2.0 μM BA and 1.2 μM 2,4-D. On average 7.1 shoots were produced per callus mass cultured on MS medium supplemented with 2.5 μM TDZ, 9.0 μM BA and 1.2 μM naphthaleneacetic acid (NAA). Regenerated shoots were transferred to MS medium supplemented with 2.5 μM TDZ, to produce multiple shoots. In vitro cultured plantlets readily acclimatized to greenhouse conditions, showing 100% survival rates in a sphagnum, perlite and sand (1:1:1) medium. These plants were transplanted into pots or planted in the field. The ex vitro acclimated plants grew vigorously and produced showy inflorescences 5–6 months after planting. The high-frequency of shoot multiplication and rapid flowering of tissue-cultured plants indicate that C. soloensis has great potential in the floricultural market.     

In vitro germination and plantlet establishment of Labisia pumila (Bl.) F. Vill.

In vitro seeds germination and plantlet establishment of Labisia pumila were studied in this report. The seeds obtained from the mature fruits of L. pumila were sterilized and cultured on Murashige and Skoog (MS) solid media supplemented with 1–3 μM of 6-benzylaminopurine (BAP) and 3% (w/v) sucrose. The presence of BAP in the medium significantly affects seeds germination. High percentage of seeds germination (up to 90%) was successfully achieved after 2 weeks of culture on medium supplemented with 2 μM BAP. Up to 70% of explants produced shoots through direct regeneration from newly emerged epicotyls after 5 weeks of culture. The average of 8.1 ± 1.0 shoots per explant obtained on media treated with 2 μM BAP. Seedlings were further transferred to growth media fortified with different types of cytokinin. Result observed after 12 weeks showed that medium supplemented with 1 μM zeatin (ZEA) promote the highest growth with an average of 2.9 ± 1.0 cm shoot length and 7.7 ± 3.2 leaves per explant after 12 weeks. In addition, medium added with 2 μM BAP and supplemented with 3–4% (w/v) of sucrose promote the best growth i.e., 3.0 ± 0.6 shoots per explant, 2.27 ± 0.2 cm length and 4.3 ± 0.5 leaves per explant.     

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.     

In vitro propagation of a grape rootstock,deGrasset (Vitis champinii Planch.): Effects of medium compositions and plant growth regulators

The aim of the present study was to develop a protocol for in vitro propagation of a grape rootstock, deGrasset, characterized by high tolerance to drought and salinity. Four medium compositions, MS, MS with 1/2 nitrates (MS-1), B5 and WPM, were tested for shoot growth from nodal explants and MS-1 medium produced significantly higher rate of shoot proliferation. MS-1 medium supplemented with 2.0 mg/L BAP was found to be optimum for culture establishment. The first subculturing on the same medium resulted in the production of 4–6, mostly short, hyperhydrated shoots per explant. For subsequent subcultures, a reduced concentration of BAP (1.0 mg/L) was used to prevent hyperhydricity, and that resulted in distinct individual shoot elongation. Three plant growth regulators, BAP, ZEA and TDZ, were tested for further shoot proliferation and BAP at 1.0 mg/L added to MS-1 medium displayed the highest proliferation rate (4.75 new explants per explant inoculated, in 6 weeks). For in vitro rooting of shoots, IAA at 0.2 mg/L was found to be optimum to produce highest response (84%) and an average number of 2.03 roots per shoot whereas use of IBA or NAA resulted in rooting with high frequency of callus formation. The acclimatized plantlets were established in soil under net house conditions with 87% success.     

Rapid in vitro multiplication and conservation of Garcinia indica: A tropical medicinal tree species

A simple and efficient method has been developed for rapid regeneration of plantlets via adventitious bud differentiation on mature seeds of Garcinia indica (Thouars) Choisy, a medicinally important facultative apomictic tropical tree species. High frequency direct shoot proliferation was induced in seed segments cultured on Murashige and Skoog's medium supplemented with cytokinins (BAP, kinetin and TDZ) alone and in combination with auxin (NAA). Amongst the various combinations used, BAP proved to be the most effective. Multiple shoots formed within 4–5 weeks of culture. The shoot forming capacity of the seeds was influenced by the BAP concentration tested (5–50 μM) and optimal response was observed at different concentrations (12.5–50 μM) in different genotypes investigated. Significant differences were recorded in terms of percent response (27.78–100%) as well as average number of shoots per explant (3.49–57.67) among the four genotypes investigated. Elongation of the induced shoots was achieved on MS basal medium containing 0.2% activated charcoal. The induction medium had a profound effect on rate of bud elongation with shoots induced on lower concentrations of BAP showing as much as four-fold elongation within 4 weeks. Proliferating shoot cultures were established by repeatedly subculturing the shoot nodes on MS medium supplemented with 5 μM BAP. Maximum rooting (91.66%) occurred in shoots cultured on half-strength MS medium supplemented with 10 μM IBA. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 15–17 weeks. For in vitro conservation, the shoot cultures were maintained on medium supplemented with 0.5 μM BAP and the subculture duration could be enhanced up to maximum of 11 months.     

In vitro morphogenesis and adventitious shoot mass regeneration of Vriesea reitzii from nodular cultures

Micropropagation systems based on nodular cultures (NCs), are considered as an intermediary in vitro morphogenetic route, diverging from regenerative systems based on organogenesis and somatic embryogenesis. The aim of this study was to establish a regenerative protocol based on the induction and development of NCs in Vriesea reitzii, an endangered bromeliad from the Atlantic forest which also has ornamental value. Additionally structural analyses were performed in order to better understand this in vitro morphogenetic route. NCs were regenerated in MSB culture medium free of PGR or supplemented with different levels of NAA alone or in combination with in combination with 2-iP. The subculture of these NCs on MSB medium supplemented with 10 μM of GA₃ promoted the synchronized shoot elongation. A regenerative efficiency of 12.4 g g⁻¹ of NCs was obtained, and this results in 5300 microshoots after 10 weeks in culture. The structural analyses of the NCs revealed that the regenerative process occurs from the proliferation of meristematic cell groups resulting in the development of multiple shoot meristems and buds. The development of NCs leads to the formation of monopolar structures called microshoots, which evolve to elongated shoots. Intermediary features shown in NCs are consistent with their classification as an intermediary system among organogenesis and somatic embryogenesis.     

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 μM 6-benzylaminopurine (BA) plus 4.5 μM thidiazuron (TDZ), and 22.2 μM BA plus 4.5 μM TDZ, respectively. Seventy-six percent of hypocotyl segments and 24% of cotyledon segments produced adventitious shoots, with a mean number of adventitious shoots per explant of 2.7 ± 0.5 and 2.3 ± 1.3, respectively. The effect of in vitro-germinated seedling age on adventitious shoot regeneration from hypocotyl and cotyledon explants was also studied. Results showed that hypocotyl and cotyledon explants from freshly isolated embryos exhibited a higher organogenesis potential than 4–15-day-old explants. Adventitious shoots from hypocotyls and cotyledons were established as proliferating shoot cultures following transfer to MS basal medium with Gamborg B5 vitamins supplemented with 10 μM BA plus 10 μM TDZ. A high rooting percentage (73–90%) was achieved when in vitro shoots were rooted on woody plant medium (WPM) containing 4.9 μM indole-3-butyric acid (IBA) and IAA (0, 2.9, 5.7, or 8.6 μM) with a combination of 10-day dark culture period followed by a 16-h photoperiod. The highest rooting (90%) of adventitious shoots or the number of roots per shoot (3.0 ± 1.0) was obtained on WPM with 4.9 μM IBA plus 5.7 μM IAA. Rooted plants were successfully acclimatized to the greenhouse and 100% survived after overwintering in cold storage. This regeneration system using hypocotyls and cotyledons provides a foundation for Agrobacterium-mediated genetic transformation of F. pennsylvanica for resistance to the emerald ash borer.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

High frequency plant regeneration through adventitious multiple shoot organogenesis in epicotyl explants of Indian gooseberry (Emblica officinalis Gaertn)

Shoots regenerated adventitiously on epicotyl segments from in vitro seedlings of Emblica officinalis var. ‘Kanchan’. Epicotyls derived from 2-week-old aseptic seedlings were most responsive and produced a maximum number of 303 shoots per explant in Murashige and Skoog (1962) medium (MS) augmented with 8.8 μM N⁶-benzyladenine (BA) + 1.425 μM indole-3-acetic acid (IAA). Shoots readily elongated in MS lacking growth regulators and rooted in half-salt-strength MS (1/2 MS) supplemented with indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). The highest rooting response was recorded in 1/2 MS containing 14.7 μM IBA. Plantlets were acclimatized inside the green house and 80% of the plantlets survived on transfer to garden soil.     

Micropropagation of the Brazilian endemic bromeliad Vriesea reitzii trough nodule clusters culture

Vriesea reitzii Leme & Costa is an endemic bromeliad from the Atlantic Forest in South Brazil. The devastation of this biome threatens the extinction of this species that besides its role in the ecosystem has an ornamental value. Tissue culture techniques are important tools for the mass propagation of threatened bromeliad species. In the present work we established an in vitro regenerative protocol for the large-scale propagation, and improvement of this species. Young basal leaves used as explants showed 90.6% induction rate of nodule clusters in MS culture medium supplemented with 20.0 μM 2,4-D and 1.0 μM Kin. The subculture of these nodule clusters to MS medium with BAP, Kin and 2-iP resulted in a regeneration rate of 60 shoots/g nodule clusters. Subsequent subculture to MS media supplemented with 2.5 μM 2-iP and 0.5 μM NAA and then to MS medium free of PGR enhanced the full development of plantlets. Plantlets longer than 3 cm were successfully acclimatized showing a survival rate of 90%.     

Seed germination and tissue culture of Cymbidium giganteum Wall. ex Lindl.

Efficient protocols were established for in vitro seed germination, neo-formation of secondary (2°) protocorms from primary (1°) protocorms and multiple shoot buds and protocorm-like body (PLB) induction from pseudo-stem segments of in vitro-raised seedlings of Cymbidium giganteum. Four nutrient media, namely Murashige and Skoog (MS), Phytamax (PM), Mitra et al. (M), and Knudson ‘C’ (KC) were evaluated for seed germination and early protocorm development. In addition, the effects of peptone, activated charcoal (AC) and two plant growth regulators [6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D)] were also studied. Both M and PM supplemented with 2.0 g l⁻¹ peptone or 1.0 mg l⁻¹ BAP resulted in ∼100% seed germination. Media supplemented with 2.0 g l⁻¹ AC could effectively induce large protocorms (1.6 ± 0.1 mm in diameter). Neo-formation of 2° protocorms from 1° protocorms was achieved in liquid and agar-solidified PM medium fortified with different concentrations and combinations of auxins (α-naphthalene acetic acid (NAA) and 2,4-D) and cytokinins [BAP and kinetin (KN)]. The highest number of 2° protocorms was obtained in liquid medium (10.7 ± 0.9/1° protocorm) supplemented with 2.0 mg l⁻¹ BAP + 1.0 mg l⁻¹ NAA. Although protocorms proliferated profusely in liquid medium, these did not develop further unless transferred to agar-solidified medium within 6–8 weeks. Multiple shoot buds and PLBs were induced from pseudo-stem segments on agar-solidified PM medium fortified with different concentrations and combinations of BAP and NAA and the maximum number of PLBs (6.00 ± 0.20) was recorded when BAP and NAA were applied at 2.0 mg l⁻¹ each. A solid root system was induced from PLBs and shoot buds when these were transferred to half-strength PM or M media fortified with 0.5 mg l⁻¹ indole-3-acetic acid. Well-rooted plants were transferred to the greenhouse with 95% survival.     

Development of a regeneration protocol through indirect organogenesis in prickly pear cactus (Opuntia ficus-indica (L.) Mill)

An indirect organogenesis regeneration protocol for Opuntia ficus-indica (L.) Mill var “Blanco sin Espinas” is described. One centimeter square cladode explants sections from previously micropropagated prickly pear plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 20 different combinations of 2,4-dichlorophenoxy acetic acid (2,4-D) and benzyladenine (BA). The best calli induction and regeneration response were observed when 2.26 μM 2,4-D and 2.21 μM BA combination was applied to the nopal explants. Regenerating calli was capable of forming new buds when transferred to MS basal medium supplemented with 0.5 μM BA (proliferation medium). Shoot elongation and rooting were achieved on MS medium without plant growth regulators. Excellent acclimatization to greenhouse conditions was observed for all transferred plantlets. By this procedure no morphological differences were observed between the regenerated and mother plants. This protocol may be also utilized to carry out plant regeneration after genetic transformation, in order to develop transformed plants without the presence of chimeric zones.     

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.     

6-Benzylamino purine stimulates in vitro shoot organogenesis in Eucalyptus erythronema, E. stricklandii and their interspecific hybrids

In vitro bud and shoot organogenesis was investigated for the ornamental plants Eucalyptus erythronema var. erythronema, E. stricklandii and their interspecific hybrids cv. ‘Urrbrae Gem’ and ‘Hybrid 2.5’ by using 0.0, 0.1, 0.25, 0.5 or 1.0 μM BAP on apex and leaf explants. Callus developed on all explants and increased with all concentrations of BAP without significant differences between BAP concentrations. Buds formed on apex and leaf explants of E. erythronema and E. cv. ‘Urrbrae Gem’ especially with 1.0 μM BAP, but these buds rarely developed into shoots. Bud clusters formed on E. erythronema and E. cv. ‘Urrbrae Gem’ apex and leaf explants whereas E. stricklandii and ‘Hybrid 2.5’ produced fewer, individual buds on the explant. Shoots regenerated from apex explants of all genotypes with all levels of BAP, whereas few shoots of any genotype regenerated from leaf explants regardless of the number of buds formed. Shoots from apex explants could be multiplied successfully. Light microscopy showed meristems developed within the callus, and at the callus and bud surfaces. However, few shoots developed considering the level of bud and meristem formation. This report is the first for successful shoot organogenesis and multiplication in an ornamental eucalypt.     

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.     

Direct shoot organogenesis on hypocotyl explants from in vitro germinated seedlings of Psidium guajava L. cv. Allahabad Safeda

Multiple shoots were generated via direct organogenesis on hypocotyl segments excised from in vitro germinated seedlings (45-day-old) of Psidium guajava L. cv. Allahabad Safeda. Modified basal Murashige and Skoog (MMS) medium supplemented with 6-benzylaminopurine (BAP), zeatin or thidiazuron with or without α-naphthalene acetic acid (NAA) were tried. Thidiazuron (0.1 μM) along with 0.54 μM NAA gave the highest response (44.6%) with the regeneration of 3.6 shoots per original explants. These shoots upon subculture gave rise to about 5.0 shootlets per explant in shoot proliferation medium, i.e. MMS supplemented with 2.22 μM BAP + 2.32 μM kinetin. The regenerated microshoots were elongated using a quick dip of gibberellic acid (GA₃; 1.44 mM) prior to culture on MMS medium supplemented with 0.88 μM BAP and adenine sulphate (54.29 μM) for 2 weeks. Rooting of microshoots was achieved best on half strength MMS medium supplemented with 4.90 μM indole-3-butyric acid along with 100 mg l⁻¹ activated charcoal.