Effect of Agar, MS Medium Strength, Sucrose and Polyamines on in vitro Rooting of Syzygium Alternifolium

This paper describes the effect of agar, MS basal medium strength, sucrose and polyamines on the in vitro rooting of Syzygium alternifolium realized by a two step procedure involving root initiation (RI) and root elongation (RE). RI was carried out on solidified MS medium supplemented with 1.0 mg dm⁻³ indole-3-butyric acid (IBA) for 3 weeks, and RE following transfer to half-strength MS medium devoid of growth regulators for another 3 weeks. Agar and MS basal medium concentrations played important role on rooting response as well as on health of rooted shoots. Sucrose concentration was positively correlated with the rooting percentage, root number per shoot and root length. The combination of polyamines and 1.0 mg dm⁻³ IBA increases rooting percentage compared to media containing only 1.0 mg dm⁻³ IBA. Optimum rooting was attained with half-strength MS medium containing 1.0 mg dm⁻³ IBA, 2 % sucrose, 10 μM spermine and 0.8 % agar. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of CuSO4 for establishing in vitro culture,and the role nitrogen and iron sources in in vitro multiplication of Corylus avellana L. cv. Tonda Gentile Romana

Abstract

In the present work, we have used copper sulphate (CuSO₄·5H₂O) enriched medium for effective control of visible and latent contamination. Among the different concentrations used, 1.25–2.5?mg/L resulted the most appropriate. In addition, the role of different nitrogen source and concentrations (NH₄NO₃ and KNO₃), as different iron source (FeEDTA and FeEDDHA) has been investigated in the proliferation and rooting phases of European hazelnut (cv. Tonda Gentile Romana). The normal concentration of nitrogen present in Murashige and Skoog medium is too high for hazelnut micropropagation cv. Tonda Gentile Romana. A reduction of total nitrogen, accompanied by a reduction of ammonium forms, resulted in a better quality of the shoots. Similar results have been obtained when the common iron source FeEDTA has been replaced by the same concentration of FeEDDHA. An increase in rooting occurs when the amount of nitrogen was reduced in the rooting medium, particularly when the NH₄NO₃ was not present.     

Multiple Shoot Induction from Cotyledonary Node Explants of Terminalia chebula

A protocol for multiple shoot induction from cotyledonary node explants of Terminalia chebula Retz. has been developed. Germination frequency of embryos (up to 100 %) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^–3 gibberellic acid (GA₃). Maximum number of shoots (6.4 shoots per cotyledonary node) was obtained on half-strength MS + 0.3 mg dm^–3 GA₃+ 1.0 mg dm^–3 indole-3-butyric acid (IBA) + 10.0 mg dm^–3 benzylaminopurine (BAP) after 4 weeks of culture. When the cotyledonary nodes along with the axillary shoot buds were allowed to grow in the same medium upto 19.2 shoots were obtained after 8 – 9 weeks. Best rooting (100 %, 5.5 roots per shoot) was observed when shoots were excised and transferred to half-strength MS medium containing 1.0 mg dm^–3 IBA + 1 % mannitol and 1.5 % sucrose. Survival of rooted plants in vivo was low (35 – 40 %) when they were directly transferred to soil in glasshouse. However, transfer to soil with MS nutrients and 1.0 mg dm^–3 IBA in culture room for a minimum duration of 2 weeks increased the survival percentage of plants to 100 %.     

Indole-3-butyric acid and myo-inositol impacts on in vitro rooting of the cherry rootstocks CAB-6P and Gisela 6

The present study investigates the effects of indole-3-butyric acid (IBA) alone and in combination with myo-inositol on in vitro rooting and biochemical responses in the cherry rootstocks CAB-6P (Prunus cerasus L.) and Gisela 6 (Prunus cerasus × Prunus canescens). For the CAB-6P rootstock, the best results for root number (6.31), fresh mass (FM), dry mass (DM), and rooting percentage (100 %) were obtained on Murashige and Skoog (MS) medium with 2 mg dm^?3 IBA and maximum root length (30.57 mm) was obtained at 1 mg dm^?3 IBA. Myo-inositol suppressed the positive effects of IBA on root length. In the Gisela 6 explants, the inclusion of 2 mg dm^?3 IBA together with 0.5 mg dm^?3 of myo-inositol in the culture medium significantly enhanced root number (9.91) and root FM and DM. The root length was maximum in the combination of the lowest IBA and myo-inositol concentrations (0.5 mg dm^?3). The rooting percentage was the greatest (100 %) with the application of 1 mg dm^?3 IBA alone. In both explants, the application of IBA alone or in combination with myo-inositol resulted in a lower leaf proline content in comparison with the control (without growth regulators). The maximum leaf chlorophyll content was at 1 mg dm^?3 IBA in the CAB-6P whereas at 2 mg dm^?3 IBA and 1 mg dm^?3 myo-inositol in Gisela 6. Addition of myo-inositol mostly increased sugar content in comparison with control or IBA alone in both rootstocks.     

Shoot Regeneration from Immature Cotyledons of Cicer Arietinum

Shoot regeneration was achieved from immature cotyledons of five chickpea (Cicer arietinum L.) genotypes: C235, ICC4971, ICC11531, ICC12257 and ICC12873. The cotyledons cultured on Murashige and Skoog (MS) medium supplemented with 3 or 5 mg dm^–3 zeatin with or without 0.04 mg dm^–3 indole acetic acid (IAA) showed formation of cotyledon like structures (CLS) at their proximal ends. Subsequently, shoot regeneration took place in some of the CLS forming explants. CLS were also formed in cotyledons cultured on MS + 0.2 – 1 mg dm^–3 thidiazuron (TDZ); direct shoot regeneration was observed in cotyledons cultured on 1 mg dm^–3 TDZ. The shoot buds elongated on media containing indole butyric acid (IBA), benzylaminopurine (BAP) and gibberellic acid (GA₃). Complete plantlets were obtained by rooting of shoots following pulse treatment with 200 mg dm^–3 IBA for 5 min and culture on growth regulator free half-strength MS medium.     

Importance of the iron chelate formula for micropropagation of Rosa hybrida L. ‘Moneyway’

In vitro propagation of the rose rootstock Moneyway was investigated on the following media: Murashige and Skoog (MS), Quoirin and Lepoivre (QL) and Woody Plant (WP). Growth, which was measured as length of shoots after a 6-week period, was faster on MS and QL than on WP. In spite of the better growth, chlorosis of newly formed leaves occurred from the third week on and was correlated with a lower chlorophyll content of shoots. Replacement of FeEDTA by FeEDDHA in QL and MS resulted in the development of green shoots for more than 3 months. The occurrence of chlorosis was not pH directed since the pH of QL with FeEDTA or FeEDDHA had not changed after 6 weeks of growth. Addition of the light absorbing dye fast yellow 9 to QL with FeEDTA also resulted in green shoots with a higher chlorophyll content. It is suggested that FeEDDHA is a more photostable chelate than FeEDTA, resulting in a higher availability of iron for the rose shoots. The impact of the iron chelate formula on the micropropagation of plant species that are susceptible to iron deficiency is discussed.Abbreviations BA 6-benzyladenine, fast yellow 9-4-amino-1,1-azobenzene-3,4-disulfonic acid - FeEDTA ferric ethylenediamine tetraacetate - FeEDDHA ferric ethylenediamine di(o-hydroxyphenylacetate) - IAA indole-3-acetic acid - IBA indole-3-butyric acid - LSD least significant difference - NAA -naphthaleneacetic acid - P probability     

Soluble sugar,starch and phenolic status during rooting of easy- and difficult-to-root magnolia cultivars

The aim of the study was to determine the effect of indole-3-butyric acid (IBA) and exogenous sucrose concentrations on in vitro rooting, soluble sugar, starch and phenolic production, and ex vitro survival of four magnolia cultivars. There was a significant linear increase in rooting of most magnolia genotypes with an increase in IBA concentration in the medium from 1 to 6 mg L^?1. A further increase of IBA concentration to 10 mg L^?1 decreased (‘Elizabeth’, ‘Burgundy’) or had no effect on rooting frequency (‘Spectrum’). The effect of IBA on rooting of magnolia shoots was modified by sucrose supply. The three out of four magnolia cultivars showed the highest rooting efficiency in the presence of 6 mg L^?1 IBA and 30 g L^?1 of sucrose. Generally, decreasing and increasing the sucrose supply from 30 g L^?1 significantly lowered the rooting frequency. In ‘Yellow Bird’, sucrose at 40 g L^?1 totally blocked root formation. It has been found that the poor rooting ability of ‘Yellow Bird’ coincided with a low soluble sugar content, and high production of starch and phenolics in the shoot bases during the whole rooting period as compared to easy-to-root cultivars. After 5 weeks of the growth on IBA medium, rooted and unrooted shoots were transferred to ex vitro conditions. Both types of shoots showed a high survival and rooting rate (85.4–100%), but they differed in their growth activity and quality. Sucrose concentration in the rooting medium had a post-effect on ex vitro root formation and survival of magnolia plantlets. Ex vitro establishment (13.3%) of recalcitrant ‘Yellow Bird’ was obtained only when the shoots were taken from rooting medium containing the lowest level of sucrose (20 g L^?1).

     

In vitro regeneration in Sarcostemma acidum (Roxb.) –an important medicinal plant of semi-arid ecosystem of Rajasthan, India

An efficient regeneration protocol for Sarcostemma acidum – an important medicinal plant has been established. Callus initiated from nodal explant on MS medium with 2.0 mg?L^?1 of NAA + additives. Callus initiated was subcultured on MS medium containing various concentrations of NAA or 2,4-D. Out of these combinations, MS medium +1.0 mg?L^?1 of NAA + additives was found to be effective for the multiplication of callus. Subculture was done after an interval of 20–22 days. For differentiation of callus BAP or Kinetin alone was found to be less effective. Maximum frequency of shoot regeneration recorded on MS medium +1.0 mg?L^?1 of BAP?+?0.5 mg?L^?1 of Kinetin and 0.1 mg?L^?1 of NAA + additives. The in vitro differentiated shoots were excised and inoculated on 1/4 strength MS medium +2.0 mg?L^?1 of IBA?+?0.02 % activated charcoal for in vitro rooting. Maximum response (90 %) was recorded on this medium. In vitro differentiated shoots were inoculated on autoclaved soilrite® after treatment with root inducing auxins. Ex vitro rooting in this plant species has been reported for the first time. Eighty five percent of the shoots rooted under ex vitro conditions. Both in vitro and ex vitro rooted plantlets were hardened in a green house.     

In vitro propagation ofBoswellia serrata Roxb

Anin vitro procedure for large scale multiplication ofBoswellia serrata Roxb. has been developed using cotyledonary node segments. In average 4 shoots per node were obtained on Murashige and Skoog’s (MS) medium containing 0.5 mg dm^?3 6-benzylaminopurine (BAP) and 0.05 mg dm^?3 napthaleneacetic acid (NAA) within 22 d. By repeated subculture technique 90–100 shoots per node could be obtained after 88 d of initial culture. Shoots could be rooted on MS medium containing 1/4 salts, 1% saccharose, and a combination of 0.5 mg dm^?3 indole-3-butyric acid (IBA) and 0.25 mg dm^?3 indole-3-acetic acid (IAA). Addition of antioxidants like polyvinylpyrrolidone (PVP-50 mg dm^?3) and ascorbic acid (100 mg dm^?3) in both multiplication and rooting media prevented browning of cultures. Approximately 80% of shoots rooted within 8–10 d. Rooted plantlets were kept for 15 d in culture bottles containing Soilrite^TM irrigated with a nutrient solution containing 1/4 MS salts and finally transferred to pots containing soil: Soilrite^TM (1∶1), mixture with 70% transplantation success.     

Thidiazuron-induced Shoot Multiplication and Plant Regeneration in Bamboo (Dendrocalamus strictus Nees)

Thidiazuron (TDZ) stimulated shoot proliferation from different seedling explants (i.e., shoot, basal node, node and apical segment) of bamboo (Dendrocalamus strictus) when incorporated in half-strength Murashige and Skoog (MS) medium having 2% (w/v) sucrose. All the concentrations of TDZ (0.01 to 1.0 mg l^?1) tried were effective in shoot proliferation. Maximum shoots (14.8 ± 1.0) were obtained from the shoot explants cultured in 0.5 mg l^?1 TDZ supplemented halfstrength MS liquid medium for 21 days and subsequently transferred to the same medium devoid of TDZ. The longer culture period (i.e. 28 and 35 days) in TDZ medium caused reduction in shoot proliferation. The shoots regenerated with lower concentrations of TDZ treatment (i.e. 0.01 to 0.1 mg l^?1) rooted in half-strength MS liquid medium. The shoots formed with 0.5 mg l^?1 TDZ treatment did not root in basal medium and required auxin supplementation in the medium for rooting and about 55% shoots produced roots in 1.0 mg l^?1 IBA supplemented medium. The shoots formed with 1.0 mg l^?1 TDZ did not root even after auxin treatment. The well rooted shoots transplanted to plastic pots filled with sand and garden soil (1:1) mixture showed 98% establishment.     

Identification of somaclonal variants in proliferating shoot cultures of Senecio cruentus cv. Tokyo Daruma

A protocol for in vitro propagation of cineraria (Senecio cruentus) was developed. The highest frequency of shoot proliferation was obtained from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with 2.0?mg L^?1 6-benzyladenine (BA) and 0.5?mg L^?1 ??-naphthalene acetic acid (NAA), with a mean number of 14 shoots per explant. A high concentration of BA (4.0?mg L^?1) and repeated subcultures resulted in hyperhydric shoots. Decreasing the BA concentration to 1.0?mg L^?1 in the culture medium eliminated hyperhydricity. The concentration of ammonium nitrate (NH₄NO₃) and temperature had marked effects on somaclonal variation. Variation was observed when the cultures were maintained at 15?°C but not at 25?°C. Variants with blue-colored leaves and stems were identified; whereas, normal plants maintained their green-colored leaves and stems. The highest frequency of variation (67.5?%), with a mean number of 3.0 variant shoots per explants, was obtained on shoot proliferation medium (MS?+?2.0?mg L^?1 BA and 0.5?mg L^?1 NAA) devoid of NH₄NO₃. The best rooting (100?%), with the highest number of roots per shoot (10.8) and the greatest root length (6.8?cm) was obtained on medium supplemented with 0.1?mg L^?1 NAA. In vitro-grown plantlets were successfully acclimatized in a greenhouse, and transferred to the field.     

In Vitro Multiplication of Paedaria foetida L — A Rare Medicinal Plant

Paedaria foetida L, which has great medicinal value is facing danger of extinction. For its conservation, in vitro multiplication may prove one of the best techniques. Micropropagation of P. foetida has been achieved through the culture of nodal explants. The explants produced shoots on MS medium with 0.8% agar. This medium, however, caused high degree of browning of the explants and death of sprouted shoots. Agar free liquid MS medium with polyvinylpyrrolidone (PVP) proved better. Maximum shoot proliferation, free from callus and vitrification but with poor rooting could be obtained in liquid MS medium with PVP (0.8%), NAA (0.5 mg l^?1) and BA (2.0 mg l^?1). The best rooting occurred on semisolid MS medium containing 0.8% agar and 0.5 mg l^?1 IBA.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Rapid micropropagation of mature wild cherry

Explants taken from the mature vigorous tree of wild cherry (Prunus avium L.) were assayed for their organogenic capacity under various phytohormonal treatments. The highest rate of adventitious shoot multiplication was recorded at a combination of 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ thidiazuron (6.83 shoots per explant). No differences in multiplication rates were found among media supplemented with BAP, BAP + α-naphthaleneacetic acid (NAA) or BAP + indole-3-butyric acid (IBA). Shoot elongation was significantly affected by the concentration of BAP, regardless of auxin addition to medium. Up to 73 % of microshoots rooted after using 0.3 mg dm⁻³ IBA, otherwise the adventitious rooting occurred at reasonable frequencies in all auxin treatments. Regenerated plantlets were successfully hardened ex vitro and continued to grow after the transfer to soil. No morphological aberrations were observed in the regenerates.     

Micropropagation of <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis>

An efficient protocol for micropropagation of Harpagophytum procumbens DC., an endangered African medicinal plant, was developed. Maximum shoot multiplication without callus was obtained from nodal explants cultured on Murashige and Skoog (MS) basal salts plus Gamborg’s (B₅) vitamins supplemented with 0.1 mg dm⁻³ indole-3-acetic acid and 5.0 mg dm⁻³ kinetin. The shoots were subsequently subcultured every 3 weeks on the same medium. Detached axillary shoots were transferred to MS basal salts plus B₅ vitamins supplemented with various concentrations of α-naphthalene-acetic acid or indole-3-butyric acid (IBA), ranging from 0.5 to 2.5 mg dm⁻³ and 100 % rooting and optimal subsequent acclimatization was achieved on 1.0 mg dm⁻³ IBA. After 4 weeks of culture, the rooted shoots (>5 cm) were planted in pots containing peat, vermiculite and bark (2:1:1), covered with plastic domes and maintained at 25 °C for 2 weeks before being transferred to a glasshouse. Plant survival was about 40 %.     

In Vitro Propagation of Endangered Temperate Himalayan Medicinal Herb Picrorhiza kurroa Royle ex Benth using Leaf Explants and Nodal Segments

The present study describes the micropropagation of Picrorhiza kurroa, (commonly known as kutki) an endangered medicinal herb of the temperate Himalayas and a source of hepatoprotective picrosides. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segments and leaf tissue. For shoot regeneration, the hormone combinations kinetin (2.0 mg l^?1) and Kinetin + Indole-3-butyric acid (IBA) (2.0 mg l^?1 + 0.50 mg l^?1) with leaf explant was found superior. Interestingly, the basal MS medium gave 99.94 % response (direct proliferation) with nodal explant. The medium supplemented with IBA (1.0 mg ^?1) was found best for rooting of regenerated shoots. Nodal segments plated on the medium supplemented with TDZ + IBA (0.11 mg ^?1 + 0.50 mg ^?1) formed somatic embryos, however further regeneration could not be achieved. The in vitro raised plantlets were hardened and successfully established in the glass house conditions.     

Tissue Culture Propagation of Elite Plant of Aloe vera Linn

Micropropagation protocol for an elite selection of Aloe vera syn A. barbadensis through enhanced axillary branching was standardized. Murashige and Skoog medium containing 1 mg l^?1 BA and 0.2 mg l^-1 IBA gave highest multiplication. Citric acid at 10mg l^-1 and liquid medium improved the shoot multiplication. Hundred per cent microshoots produced rooted plantlets within 15 days of culture on hormone-free agar medium. Liquid medium during rooting stage decreased the number of shoots showing rooting response. The plants were successfully transferred in the soil and were morphologically similar to mother plants.     

In Vitro Method for Propagation of Centella asiatica (L) Urban by Shoot Tip Culture

A rapid clonal propagation system has been developed for the medicinally important herb Centella asiatica (L) Urban by shoot tip (2–3 cm long) culture. The shoot tips isolated from mature plants were inoculated on MS medium incorporated with BA alone or in combination with NAA and Kn. The optimum number of shoots (3.38) with optimum number of leaves per shoot (4.25) were attained on MS medium supplemented with 4.0 mg l^?1 BA and 0.1 mg l^?1 NAA. On transferring the microshoots on full strength MS medium supplemented with various concentrations of IBA (1.0-3.0 mg l^?1) and NAA (0.5-2.0 mg l^?1), profuse rooting (46.8 per shoot) was obtained in MS basal medium with 2.0 mg l^?1 IBA with root length of 19.7 cm. Well rooted plantlets were acclimatized successfully by adjusting the temperature and humidity for 3–4 weeks after transfer to pots filled with sterilized vermiculite soil: sand (1:1)mixture. This micropropgation protocol could be useful for raising a stock of genetically homogenous material for field cultivation within a very short period.     

In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.     

Micropropagation of Anethum graveolens L Through Axillary Shoot Proliferation

A protocol is described for rapid and large-scale in vitro propagation of Anethum graveolens by enhanced axillary shoot induction that was dependent on BAP supply. The synergistic combination of 0.5 mg l^?1 BAP and 0.1 mg l^?1 IBA induced 100% shoot formation as well as shoot number (6.6 ± 0.48 per explant). Subculturing of shoot tips of in vitro plants on multiplication medium enabled continuous production of healthy shoots with similar frequency. Rooting of shoots was achieved on a medium with 1mg l^?1 IBA and 0.5 mg l^?1 Kn. Micropropagated plants established in garden were uniform and identical to the donor plant with respect to morphological and cytological characteristics.