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.     

Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor)

Lanzhou lily (Lilium davidii var. unicolor) is one of the best lilies which are edible in China but the efficient shoot regeneration system has not been developed. The purpose of the present study is to establish an efficient and reproducible protocol for induction of shoots in vitro from L. davidii var. unicolor leaves. Shoot regeneration from in vitro cultured leaves of L. davidii var. unicolor was tested on the 26 media based on NN [Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen grains. Science 163, 85–87] basal medium, containing different concentrations of thidiazuron (TDZ) in combination with different concentrations of α-naphthaleneacetic acid (NAA). Shoot organogenesis occurred directly from the leaves without forming callus. Shoot regeneration mainly occurred from the cuts across the midvein and the base of the leaf explants. The highest frequency of regeneration (93.3%) and the largest number of shoots per leaf (3.83) were obtained on NN basal medium supplemented with 0.5 mg l⁻¹ TDZ and 1.0 mg l⁻¹ NAA. All the regenerated shoots formed complete plantlets on half-strength MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] basal medium containing 0.1–0.5 mg l⁻¹ indole-3-butyric acid (IBA) with in 30 days, and 92% of the regenerated plantlets survived in the soil. This study will be useful for Agrobacterium-mediated transformation and exploitation of somaclonal variation of Lanzhou lily.     

Effect of genotype,explant size,position, and culture medium on shoot generation of Gerbera jamesonii by receptacle transverse thin cell layer culture

An unique procedure for the mass shoot propagation of Gerbera using receptacle transverse thin cell layer (tTCL) culture procedure was developed. Genotype, flower bud age, explant size, position of receptacle tTCLs and culture media were found to affect the success of culture. Ten interspecific crosses of Gerbera showed different shoot regeneration rates and callus induction via receptacle tTCL culture, all of which had shoot regeneration rates higher than 57%. Flower buds collected on the 10th day resulted in 91% shoot regeneration after 6 weeks of culture on basal MS medium [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 475–497] supplemented with 0.02 mg l⁻¹ thidiazuron (TDZ), 0.8 mg l⁻¹ adenine and 10% (v/v) coconut water (CW). This was significantly higher than those from flower buds on the 7th and 14th days (22% and 54%), respectively. Shoot regeneration rate was the highest (94–100%) in the middle layers of the receptacle. For mass shoot propagation, shoot clusters were subcultured on half-strength MS medium supplemented with 0.5 mg l⁻¹ indole-3-butyric acid (IBA), 0.5 mg l⁻¹ 6-benzyladenine (BA) and 2.0 mg l⁻¹ kinetin after every 4 weeks. Plantlets formed when single shoots were cultured on half-strength MS medium containing 1 mg l⁻¹ IBA. All plantlets acclimatized well in the greenhouse.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia L.

An effective protocol was developed for in vitro regeneration of Psoralea corylifolia through enriched cotton moistened-liquid (CML) and solid culture systems. Prolific adventitious shoot buds were achieved from hypocotyl explants of 2-week-old cultures on enriched CML Phillips and Collins (L2) medium supplemented with different concentrations and combinations of thidiazuron (TDZ), benzyladenine (BA), kinetin (KIN), naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and bavistin (BVN). Combination of 2 μM TDZ, 0.5 μM BA, 100 mg l⁻¹ BVN and 2 μM NAA produced a greater number of adventitious shoots per explant (93.5) when transferred to half-strength enriched solid L2 medium. Regenerated shoots (40–50 mm in length) were exposed simultaneously for rooting as well as hardening in moistened (1/8-L2 basal salt solution with 5 μM IBA and 100 mg l⁻¹ BVN) soil mixture and vermiculite (3:1, v/v). The plants were subsequently established in the field. The survival percentage differed with seasonal variations.     

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.     

Direct plant regeneration from mature leaf explants of pistachio, Pistacia vera L.

A protocol was developed for direct shoot and plantlet regeneration from in vitro regenerated leaf explants of male Pistacia vera L. cv. ‘Atl?’. Leaves excised from axenic shoot cultures of pistachio were used to induce organogenesis on a Murashige and Skoog (MS) medium with Gamborg vitamins supplemented with combinations of different concentrations of BAP and IAA. The highest adventitious shoot regeneration in 35% of the explants, with the number of shoots ranging from 2 to 3 per explant, occurred in the explants cultured during the establishment phase in the medium with 1 mg l⁻¹ IAA and 2 mg l⁻¹ BAP. For shoot multiplication, the highest number of new microshoot/explants (5.76) was obtained in a culture medium supplemented with 1 mg l⁻¹ BAP, but it was not significantly different from the number obtained at 2 mg l⁻¹ BAP. A high rooting frequency (84%) for microshoots was recorded on a medium supplemented with 2 mg l⁻¹ IBA. In vitro rooted plantlets were transferred to pots filled with a mixture of soil, sand and peat (1:1:1). They were weaned in a growth room and finally moved to a greenhouse. This protocol could be utilized for in vitro clonal propagation of this economically important plant.     

Micropropagation of the bromeliad Guzmania ‘Hilda’ via organogenesis and the effect of α-naphthaleneacetic acid on plantlet elongation

This study established a highly effective micropropagation system to obtain good plantlet proliferation from floral organs via callus induction and bud differentiation in Guzmania ‘Hilda’ bromeliad. The best frequencies of organogenic callus formation (20% in petal and 35% in ovary explants) were obtained on media containing a combination of 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) with 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA) and 1.5 mg l⁻¹ 2,4-D with 0.5 mg l⁻¹ NAA, respectively. Organogenic calli were cultured on medium with 1.0 mg l⁻¹ NAA and 0.5 mg l⁻¹ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) induce the differentiation and regeneration of adventitious buds into plantlets. When the plantlets were cultured in a medium with optimum NAA concentration (0.5–1.0 mg l⁻¹) significant improvement in regeneration and elongation was achieved within one month. This overcame the difficulty of delayed elongation in Guzmania plantlets. More than 99% of the regenerated and acclimatized plantlets developed to the flowering stage.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO₃ on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l⁻¹, 6-BA at 3.0 mg l⁻¹, AgNO₃ at 4.0 mg l⁻¹ and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l⁻¹, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.     

Comparison of shoot induction ability of different explants in herbaceous peony (Paeonia lactiflora Pall.)

Shoot induction ability of explants of herbaceous peony was investigated in semisolid MS medium containing BA, TDZ and GA₃. Callus was readily induced from stem without node and petiole explants within 2 days of culture but failed to generate shoots. Adventitious shoots were successfully produced from meristematic regions only: bud eyes on nodal stem sections, and junctions of petioles and petiolules. No shoots were induced from internode sections, petiole without junctions, or leaf sections. Nodal sections were the most efficient explants. There were up to 20 shoots in one explant generated within 20 days of culture. TDZ was more effective than BA to induce shoots. The 100% shoot induction rate was obtained in medium containing 0.1–3 mg L⁻¹ of TDZ. However, higher concentrations of TDZ inhibited shoot elongation and only large leaf clusters were produced. Combinations of BA and TDZ failed to increase shoot induction rates but caused shoots shorter. The 2–60-min pretreatment of explants with 20 mg L⁻¹ TDZ solution was very effective to induce adventitious shoots directly, but both shoot number and shoot length tended to decrease as treatment time increased. GA₃ was beneficial for shoot and stem elongation.     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

Plant regeneration of an endangered medicinal plant Hydrastis canadensis L.

Goldenseal (Hydrastis canadensis L.) is an endangered medicinal plant used to treat sore eyes and mouths, cold and flu and also as a dye. The objective of this study was to develop an efficient in vitro propagation protocol for goldenseal. Significantly more shoots (26 shoots per leaf explants) were induced on a medium containing 2.5 μM thidiazuron (TDZ) and 5.0 μM 1-naphthaleneacetic acid (NAA) than any other treatment. Sub-culturing regenerated shoots on a medium with 5.0 μM 6-benzylaminopurine (BA) induced the maximum rate of shoot multiplication. Growth of the regenerated shoots in a temporary immersion bioreactor resulted in significant increases in biomass, shoot height and shoot multiplication. The regenerated shoots from the temporary immersion bioreactor formed roots when transferred onto a medium with 1.0–2.0 μM indole-3-butyric acid (IBA). Regenerated whole plantlets were acclimatized and maintained in standard greenhouse conditions for further growth. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of this rare, medicinally important species.     

Direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis (Girard) Kuntze

The potentialities of direct somatic embryogenesis and plant regeneration from leaf explants of Limoniumsinensis var. Golden Diamond invitro were investigated. Young whole leaf and cut leaf explants when cultured on MS basal medium supplemented with each of the growth regulators N⁶-benzyladenine (BA) (0.44–2.2 μM) or thidiazuron (TDZ) (4.54 μM) alone or in combination with a fixed concentration of α-naphthalene acetic acid (NAA) (1.07 μM) produced somatic embryos directly. More than 90% of the leaf explants produced white, globular somatic embryos on BA (2.2 μM) and NAA (1.07 μM) supplemented MS basal medium within 1 week of inoculation. Most of the embryos matured further and converted after 8 weeks of culture on the same medium. Histological observation showed that the somatic embryos originated from single cells of epidermal layer of leaf. Histological evidence of formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. After conversion the plantlets were further placed on MS medium containing 0.44 μM BA and 4.5 μM IBA for better shoot and root growth. About 90% of the plantlets transferred to the mixture of soil:perlite:vermiculite (1:1:1) in small plastic pots acclimatized successfully. Of these 85.5% plants survived after transferring into earthen pots containing a mixture of soil, coarse sand and cattle manure (1:1:1) under greenhouse or shady open condition.     

Clonal propagation of Rosa clinophylla Thory. through axillary bud culture

A protocol for clonal propagation of Rosa clinophylla, a rare and endangered species but very important for breeding purposes had been standardized through in vitro axillary bud culture. Although cytokinins alone were able to induce shoot buds, but their proper growth and number could be increased only when they were used in combination with GA₃. However, there was shoot tip necrosis and leaf fall in the proliferated shoots. AgNO₃ at 58.85 μM proved effective to avoid shoot necrosis and yellowing of leaves. Activated charcoal (AC) at 250 mg l⁻¹ was found necessary at all the stages of shoot multiplication as well as rooting. Ninety percent rooting could be achieved in 1/2 MS medium supplemented with 4.92 μM IBA and 250 mg l⁻¹ AC. Rooted plantlets were hardened and transferred to the field successfully with 80% survival rate.     

In vitro shoot regeneration from immature seeds of Epimedium alpinum induced by thidiazuron and CPPU

In vitro propagation of Epimedium alpinum L. was carried out using immature seed explants. The effects of various concentrations of thidiazuron (TDZ) and 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), on the induction of organogenic callus, were evaluated. Organogenesis occurred most efficiently when explants were transiently exposed (48 h) to 20 μM CPPU or 80 μM TDZ followed by culture on hormone-free woody-plant medium (WPM). Organogenic callus consisting of white, compact clumps of tissue proliferated slowly on hormone-free WPM. To promote adventitious shoot induction, the effects of different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) were investigated. The highest per cent shoot regeneration, 66.7% of explants, and the maximum mean number of shoots, 2.6 per explant, were obtained on WPM containing 1.1 μM 2,4-D and 22 μM BA. Shoots were rooted on hormone-free WPM and well-developed plantlets were successfully transferred to soil.     

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.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

The effects of explant and cytokinin type on regeneration of Prunus microcarpa

We investigated in vitro regeneration ability of Prunus microcarpa subsp. tortusa using various explants (root, cotyledon and hypocotyl pieces) and cytokinins [benzyladenine (BA), meta-Topolin (mT) and thidiazuron (TDZ)]. Sectioned cotyledon, root and hypocotyl pieces of in vitro grown seedlings were cultured on Nas and Read Medium (NRM) containing BA (7.5, 10, 12.5, 15 or 17.5 μM), mT (2.5, 5.0, 7.5, 10 or 12.5 μM) or TDZ (2.5, 5.0, 7.5, 10 or 12.5 μM). As a measurement of morphogenetic reaction, the ratios of regenerating explants and the numbers of primary adventitious shoots per regenerating explant were analyzed. Cotyledon explants exhibited higher regeneration ratios than hypocotyl explants, and the root explants were inappropriate for regeneration. Both BA and mT were effective on shoot regeneration but higher regenerating explant ratios were obtained when BA was used. In comparison with BA and mT, the effect of TDZ on enhancing explant regeneration ability was insignificant. Mean number of adventitious shoot per regenerating explant was between 1 and 4, and regenerating explant ratios were between 0% and 77%. The practical appliacations of the results are discussed.     

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.     

Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian

The in vitro formation of newly formed adventitious buds and shoots from internodal branch segments was studied on 12-month-old plants of Citrus aurantium L. cv. Brazilian. The effects of 6-Benzyladenine (BA) and α-Naphthalene acetic acid (NAA) treatments were evaluated on adventitious bud and shoot regeneration. High rates of bud initiation and shoot development were obtained both with BA supplemented medium, in the range from 1 mg L⁻¹ to 3 mg L⁻¹, and with 0.1 mg L⁻¹ NAA supplemented medium. NAA concentrations above 1 mg L⁻¹ significantly reduced bud initiation and shoot elongation. The results obtained using different in vitro culture vessels such as Petri dishes, tubes and glass culture jars were compared. The highest adventitious bud induction was observed in Petri dishes for internodes cultured in 2 mg L⁻¹ BA supplemented medium, with 95% responsive explants forming 9.0 ± 2.4 adventitious buds. The adventitious buds observed in Petri dishes reached a maximum height of 1 mm, with no further development, while some of the adventitious shoots cultured in tubes and glass culture jars grew over 1 cm in height. A shoot regeneration gradient of the internodes collected along the branch axis was noticed, with basal ones exhibiting higher regeneration frequency.