Direct shoot regeneration from leaf explants of <Emphasis Type="Italic">Spilanthes acmella</Emphasis>

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm⁻³ 6-benzyladenine (BA) and 0.1 mg dm⁻³ α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm⁻³ BA and 1.0 mg dm⁻³ IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm⁻³ IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.     

In vitro plant regeneration from leaf explants of Ophiorrhiza japonica

An efficient in vitro plant regeneration system from leaves of Ophiorrhiza japonica Blume was established for the first time. Callus formation rate was more than 90.4 % from leaf segments on Murashige and Skoog (MS) supplemented with either α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzyladenine (BA). The highest shoot regeneration (78.9 %) was achieved on MS medium containing 2.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA, with an average of 9.4 shoots developed per leaf segment. Shoot regeneration was also improved when the leaf explants were cultured in MS basal medium supplemented with 0.5 % (m/v) polyvinylpyrrolidone (PVP). The leaf explants from seedlings with age of about 18–27 d showed the highest shoot regeneration. The regenerated shoots were rooted on half-strength basal MS medium supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA), which averagely produced 24.8 roots per shoot. The plantlets were transferred to soil, where 100 % survived after 1 month of acclimatization.     

Micropropagation of Coleus blumei from nodal segments and shoot tips

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm⁻³) and NAA (1 mg dm⁻³). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm⁻³). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.     

Micropropagation of Ulmus pumila L. from mature trees

Summary Multiple shoots were induced from nodal segments of mature trees of Ulmus pumila L. on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). Further multiple shoots were obtained from nodal segments taken from in vitro proliferated shoots when cultured in MS medium containing 0.5 mg.l^–1 BA. Rooting of the shoots was achieved on half or full strength MS medium or in MS medium supplemented with 0.1 mg.l^–1 naphtaleneacetic acid (NAA). Rooted plantlets were able to resume independent growth after a short period of acclimatization.     

<Emphasis Type="Italic">In vitro</Emphasis> multiplication of heavy metals hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

A micropropagation protocol through multiple shoot formation was developed for Thlaspi caerulescens L., one of the most important heavy metals hyperaccumulator plants. In vitro seed-derived young seedlings were used for the initiation of multiple shoots on Murashige and Skoog (MS) medium with combinations of benzylaminopurine (BA; 0.5–1.0 mg dm⁻³), naphthaleneacetic acid (NAA; 0–0.2 mg dm⁻³), gibberellic acid (GA₃; 0–1.0 mg dm⁻³) and riboflavin (0–3.0 mg dm⁻³). The maximum number of shoots was developed on medium containing 1.0 mg dm⁻³ BA and 0.2 mg dm⁻³ NAA. GA₃ (0.5 mg dm⁻³) in combination with BA significantly increased shoot length. In view of shoot numbers, shoot length and further rooting rate, the best combination was 1.0 mg dm⁻³ BA + 0.5 mg dm⁻³ GA₃ + 1.0 mg dm⁻³ riboflavin. Well-developed shoots (35–50 mm) were successfully rooted at approximately 95 % on MS medium containing 20 g dm⁻³ sucrose, 8 g dm⁻³ agar and 1.0 mg dm⁻³ indolebutyric acid. Almost all in vitro plantlets survived when transferred to pots.     

Micropropagation of Morus laevigata Wall. from mature trees

Multiple shoots were obtained from nodal explants of 10-year-old tree of Morus laevigata on Murashige and Skoog's medium supplemented with different concentrations (0.5–5.0 mg.l^–1) of benzyladenine (BA). Nodal segments taken from in vitro proliferated shoots gave further multiple shoots when cultured on the same basal medium containing 2.5 mg.l^–1 BA. Repeated subculture resulted in rapid shoot multiplication at the average rate of 6-fold per subculture. In vitro raised shoots rooted on MS medium containing 0.1 mg. l^–1 each of 3-indolebutyric acid (ISA) and -naphthaleneacetic acid (NAA). The regenerated plantlets were successfully established in soil under field conditions after a few days of indoor acclimatization.     

<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.     

Adventitious Shoot Regeneration and Micropropagation in Calendula officinalis L.

Hypocotyl, cotyledon and cotyledonary node explants of Calendula officinalis L were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of thidiazuron (TDZ), kinetin (KIN), -naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) to induce adventitious shoot regeneration and micropropagation. The highest frequency of adventitious shoot regeneration was achieved from hypocotyl and cotyledon explants on MS media supplemented with 0.75 mg dm^–3 TDZ and either 0.25 or 0.50 mg dm^–3 IBA. Efficient in vitro clonal propagation was also induced from cotyledonary nodes on a range of media supplemented with 0.75 mg dm^–3 TDZ and 0.05 mg dm^–3 NAA or 2 mg dm^–3 KIN and 1 mg dm^–3 NAA. Regenerated shoots were excised and rooted in MS medium supplemented with 1 mg dm^–3 NAA. The rooted plantlets were finally transferred to pots.     

Micropropagation of an Endangered Orchid Anoectochilus formosanus

A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of jewel orchid (Anoectochilus formosanus). Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with 1 mg dm^–3 benzyladenine or 1 – 2 mg dm^–3 thidiazuron (TDZ). Addition of activated charcoal (1 g dm^–3) to the TDZ containing medium promoted multiple shoot formation (11.1 shoots per explant). However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 medium supplemented with 2 % sucrose and 0.5 g dm^–3 activated charcoal. Rooting was induced in 100 % of the regenerated shoots in the same media. The plantlets were acclimatized and established in greenhouse.     

Thidiazuron stimulates adventitious shoot regeneration in different safflower explants

Adventitious shoot regeneration from root, hypocotyl, cotyledon and primary leaf explants of safflower (Carthamus tinctorius L.) was studied. Shoot regeneration was promoted by benzyladenine (BA) + naphthaleneacetic acid (NAA), BA + indole-3-butyric acid (IBA), kinetin + NAA and thidiazuron (TDZ) + NAA incorporated in Murashige and Skoog (MS) basal medium. High frequency of shoot regeneration and high number of shoots per regenerating explant were obtained on a wide range of TDZ + NAA combinations. Proliferated shoots were elongated in MS + 0.5 mg dm⁻³ kinetin and well-developed shoots were rooted in half strength MS + 0.5 mg dm⁻³ NAA. Rooted shoots were successfully acclimatized and established in soil.     

A simple and efficient plant regeneration system for leaf protoplasts ofNierembergia repens by inducing single shoots on the microcolonies

A simple and efficient protocol for plant regeneration from mesophyll protoplasts ofNierembergia repens is described. The protoplasts divided in modified half-strength MS (/12 MS) medium containing benzylaminopurine (BA) and a-naphthaleneacetic acid (NAA) and formed visible colonies after 2 weeks which produced single adventitious shoots 4 weeks later. Plating efficiency (11.2%), percent colony formation (0.84%), and the number of shoot-forming colonies (368/dish) were highest in /12 MS containing 0.1 mg/l BA and 0.05 mg/l NAA. However, the percentage of colonies with shoot formation was highest (31.8%) in /12 containing 0.05 mg/l BA and 0.01 mg/l NAA. Almost all of the remaining colonies (97.5%) also regenerated shoots upon transfer onto MS medium containing 0.05 mg/l BA. The shoots with 2–3 leaves readily rooted 3–5 days after insertion in /12MS lacking plant growth regulators. Regenerated plantlets were easily established in soil 50 days after protoplast isolation. All the regenerants were normal and possessed diploid chromosome numbers.Abbreviations BA Benzylaminopurine - FDA fluorescein diacetate - MES 2-(Morpholino)ethanesulfonic acid - MS Murashige and Skoog (1962) medium - /12MS half strength MS - NAA -naphthaleneacetic acid - PGR plant growth regulator     

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 %.     

An efficient in vitro propagation of Aristolochia indica

A rapid and efficient in vitro plant regeneration method was developed for Aristolochia indica. Multiple shoot formation was induced from shoot tip and nodal explants on Murashige and Skoog (MS) medium with 1 – 6 mg dm⁻³ 2-isopentenyl-adenine (2-iP) or 1 – 4 mg dm⁻³ 6-benzyladenine (BA). Maximum number of shoots were induced with 5 mg dm⁻³ 2-iP alone (about 12 – 14 shoots). Shoot differentiation occurred directly from the leaf bases as well as from the internodes when cultured on 1 – 4 mg dm⁻³ BA and 0.8 – 2 mg dm⁻³ α-naphthaleneacetic acid (NAA) containing medium. Regeneration from the callus occurred when the calli initiated on MS medium containing 0.6 – 4 mg dm⁻³ NAA in combination with 0.8 – 3 mg dm⁻³ BA were transferred to 1 – 6 mg dm⁻³ BA alone containing medium. Elongated shoots were separated and rooted in MS medium containing 1 mg dm⁻³ indole-3-butyric acid. These were then transferred to soil after gradual acclimatization.     

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.     

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.     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

High frequency plant regeneration from the cotyledonary node of common bean

An efficient regeneration system for Phaseolus vulgaris was developed from mature seeds germinated on Murashige and Skoog (MS) medium supplemented with thidiazuron or N⁶-benzylaminopurine (BA) for 6 d. Using cotyledonary nodes, multiple buds were induced on the MS medium supplemented with 5.0 mg dm⁻³ BA with the induction frequency 71.9 % after 4-week culture. The buds were then transferred onto shoot formation medium containing 1.0 mg dm⁻³ BA, 0.1 mg dm⁻³ gibberellic acid and 2.0 mg dm⁻³ silver nitrate. The addition of AgNO₃ enhanced the frequency of the shoot formation from 61.3 to 87.6 %. Root induction medium was half-strength MS medium with 0.75 mg dm⁻³ indolebutyric acid and 0.02 mg dm⁻³ BA. The average root frequency was 84.3 %. The regenerated plantlets with healthy roots grew successfully when transferred to soil. Using this system we obtained over 10 regenerated plantlets from one explant.     

Somatic organogenesis and plant regeneration in <Emphasis Type="Italic">Ricinus communis</Emphasis>

An in vitro propagation system was developed for castor-bean (Ricinus communis L. cv. TMV 6) through cotyledon derived callus cultures. The impact of different concentrations of auxins, cytokinins, additives, amino acids and sugars were evaluated for callus induction and shoot proliferation. Green compact nodular organogenic callus was obtained on the medium fortified with Murashige and Skoog (MS) salts, B5 vitamins, 2.0 mg dm⁻³ 6-benzyladenine and 0.8 mg dm⁻³ α-naphthalene acetic acid (NAA). Multiple shoot proliferation from the callus cultures was achieved on the medium with MS salts, B5 vitamins, 2.5 mg dm⁻³ thidiazuron (TDZ), 0.4 mg dm⁻³ NAA and 15 mg dm⁻³ glutamine. During multiple shoot induction the phenolic secretion was controlled by the addition of 15 mg dm⁻³ polyvinylpyrolidone. The proliferated shoots were elongated on the medium comprising MS salts, B5 vitamins, 1.5 mg dm⁻³ TDZ and 0.3 mg dm⁻³ gibberellic acid. The elongated shoots were rooted on the medium containing MS salts, B5 vitamins, 0.3 mg dm⁻³ indole-3-butyric acid and 0.6 mg dm⁻³ silver nitrate. After root induction, the plants were hardened in earthen pots containing sand, soil and vermiculite.     

Micropropagation of Sesbania rostrata from the Cotyledonary Node

Multiple shoots were induced from the cotyledonary nodes derived from seedling of Sesbania rostrata on Nitsch (1969; N) medium supplemented with various concentrations of benzyladenine (BA). 1 mg dm⁻³ BA proved to be the best, eliciting 5.8 ± 1.0 shoots per explant in 100 % cultures. The elongation of shoots was best at 2.0 mg dm⁻³ BA. The shoot proliferation capacity increased to 7.5 shoots per explant following transfer of explants to the fresh shoot multiplication medium (MS + 1.0 mg dm⁻³ BA), after an initial incubation of 30 d. To further enhance number of shoots per explant an alternative strategy of cultivation of mother explant on fresh shoot multiplication medium after excision of shoots was adopted. Following the repeated harvesting of shoots an average of 33 shoots per explant could be obtained. The in vitro regenerated shoots produced roots when transferred to half-strength MS medium supplemented with 3 % sucrose and 1 mg dm⁻³ IBA. The developed plantlets were planted in the soil and transferred to the field after an acclimatization period of 3 – 4 months. These plants produced flowers and fruits in the field and exhibited the development of prominent and more organized stem nodules as compared to the in vivo raised plants of the same age. This revised version was published online in July 2006 with corrections to the Cover Date.