Gibberellic acid regulation of adventitious shoot formation from tuber discs of potato

Summary The formation of adventitious shoots from potato tuber discs explanted onto a modified Murashige and Skoog (MS) medium containingN ⁶-benzylaminopurine (BAP) (3.0 mg/l), and α-naphthaleneacetic, acid (NAA) (0.01 mg/l), was affected by gibberellic acid (GA). The presence of GA in the explant medium was required for shoot formation and 3×10⁻¹⁰ M GA appeared optimum. However, microscopic examination of the tissue protuberances on the surface of the tuber discs from which shoots arose revealed that GA inhibited the formation of shoot meristems. Tuber discs cultured for 6 wk on MS medium containing BAP and NAA without GA did not initiate adventitious shoots that could be determined visually, but microscopic examination of the tissue protuberances revealed the presence of numerous shoot meristems. Subsequent transfer of these tuber discs to medium with GA but without BAP or NAA resulted in the formation of shoots from 100% of the recultrued dises. Thus it appears that although GA inhibits shoot meristem initiation from potato tuber discs, it is required for shoot development once meristems are initiated. This is Journal Paper 8297 of the Purdue University Agricultural Experiment Station. The research was supported by Purdue University Agricultural Experiment Station Program Improvement Funds. Potato tubers were supplied by Wm. Gehring Farms, Inc., Rensselaer, Indiana.     

Rapid clonal propagation of three mulberries,Morus cathayana HemsL,M. lhou Koiz. andM. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5–1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.Abbreviations BAP 6-Benzylaminopurine - GA ₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthalene acetic acid     

Effect of growth regulators onin vitro propagation ofFicus benjamina cv. Exotica

Stem internodes with axillary buds were excised from 5-year old trees ofFicus benjamina cv. Exotica. The effect of 6-benzylaminopurine (BAP), gibberellic acid (GA₃), indole-3-acetic acid (IAA), naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on shoot growth and proliferationin vitro was investigated. Multiple shoots were developed after 3–4 weeks from stem internodes with axillary buds incubated in Murashige and Skoog (MS) medium supplemented with phloroglucinol (PG) and BAP. Optimum shoot proliferation took place in the presence of 1.0 mg l⁻¹ BAP. Shoots obtained could be elongated in a medium with 0.5 mg l⁻¹ GA₃ prior to their rooting. The root initiation was successfully induced on MS medium either with IAA at 0.5–0.1 mg l⁻¹ or in plant growth regulator-free medium. All rooted plantlets were subsequently transferred to a peat, humus and perlite mixture in a culture room with high humidity and covered with plastic bags. After one month the plantlets were established for growing in a greenhouse. Communicated by J. TUPY     

Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.)

A highly efficient and reproducible method of in vitro propagation using meristematic explants has been developed for castor. Embryo axes and shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 0.5–10.0 mg/l of adenine, N⁶-benzyladenine (BA), kinetin (Kn), thiadiazuron (TDZ) and zeatin. TDZ (1.0–10.0 mg/l) gave the maximum number of shoots (37.8–40.0) from embryo axes, while BA (2.0 mg/l) was found superior to other cytokinins for obtaining the highest number of shoots (46.7) from the shoot apex. Adenine and Kn at all of the tested concentrations resulted in low proliferation rates from embryo axes. The carryover effect of the cytokinins was tested by subculturing proliferating shoot cultures from various media onto the medium fortified with 0.5 mg/l BA. There was no significant influence of the cytokinins on subsequent proliferation from the two explant types except for TDZ with embryo axes. The number of shoots from TDZ-habituated embryo axes ranged between 36.0 and 81.7, while it varied from 5.7 to 22.0 and 3.7 to 28.3 in axillary buds and embryo axes, respectively, on the other media. For elongation of shoots, gibberellic acid (GA₃) (0.1–1.0 mg/l) was added to the medium supplemented with 0.2–0.5 mg/l BA. Incorporation of GA₃ (0.1 mg/l) significantly enhanced the frequency of elongated shoots but drastically reduced the multiplication ability. Hence, proliferating shoot clusters were periodically transferred to the medium supplemented with 0.5 and 0.2 mg/l BA for further multiplication and elongation. Well-developed shoots were rooted on half-strength MS medium supplemented with 1.0 mg/l indole-3-butyric acid. The rooted plantlets were acclimatized with more than 60% success. Received: 17 June 1997 / Revision received: 3 September 1997 / Accepted: 20 September 1997     

Direct Shoot Bud Formation and Tuberization from Aseptically Cultured Root Tubers of Calla Lily (Zantedeschia aethiopica L)

We describe here the development of a micropropagation protocol for mass multiplication of Zantedeschia aethiopica by using root tubers as explant. The surface sterilized root tubers produced five to six shoot-buds on semi-solid Murashige and Skoog’s (MS) medium with 10.0 mg l^?1 of 6-benzylaminopurine (BAP) and additives (50.0 mg l^?1 of ascorbic acid; 25.0 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The cultures were multiplied by sub-culture of individual shoot bud produced in vitro and clumps of shoot buds generated in vitro in cultures on MS medium containing 3.0 mg l^?1 of BAP and additives. Further multiplication of propagules was achieved through tuber formation along with amplifying shoots on MS medium with 5.0 mg l^?1 of BAP. The micropropagated shoots were rooted both in vitro as well as ex vitro. Cent percent of the cloned shoots rooted in vitro within 15–18 days on hormone-free 1/2 strength MS salts with 200.0 mg l^?1 of activated charcoal. Alternatively 95–100% shoots rooted ex vitro under greenhouse conditions on soilrite after pulse-treatment with 500.0 mg l^?1 of Indole-3-butyric acid (IBA) or β-naphthoxyacetic acid (NOA) for 300 sec. The cloned plants were hardened in the greenhouse. The hardened plants were transplanted to soil for further acclimatization.     

Efficient Evergreen Plant Regeneration of <i>Cinnamomum japonicum</i> Sieb. through <i>in vitro</i> Organogenesis

Cinnamomum japonicum Sieb. is an excellent roadside tree and medicinal tree species with considerable ornamental and economic value. In this study, we successfully developed a large-scale micropropagation protocol for C. japonicum for the first time. Sterilized shoots were excised and used as explants for shoot induction on several basal media, supplemented with different concentrations of plant growth regulators (PGRs), such as Thidiazuron (TDZ), N⁶ -Benzyladenine (6-benzylaminopurine) (BA), α-naphthaleneacetic acid (NAA) and Gibberellic acid (GA₃). After comparison, the most efficient medium for shoot regeneration was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg L^–1 BA, 0.05 mg L^–1 NAA and 0.2 mg L^–1 GA₃, which resulted in an average number of induced shoots per explant and shoot length of 5.2 and 1.62 cm at 28 d, respectively. Then, elongated adventitious shoots were transferred to induce roots. 86.7% of shoots was able to root on 1/2 MS medium supplemented with 0.5 mg L^–1 NAA and 0.1 mg L^–1 BA. The earliest rooting time observed was after 21 d and the average root length was up to 3.3 cm after 28 d. Our study shows that C. japonicum can be successfully regenerated through de novo organogenesis, which lays a foundation for future transformation research on this tree.     

Plants obtained from the Khair tree (Acacia catechu Willd.) using mature nodal segments

An in vitro method for obtaining plants of Acacia catechu has been developed using nodal explants from mature `elite' trees growing in the field. Maximum shoot bud development (eight to ten) from a single explant was achieved on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) (4.0 mg/l) and α-naphthaleneacetic acid (0.5 mg/l). Addition of adenine sulphate (25.0 mg/l), ascorbic acid (20.0 mg/l) and glutamine (150.0 mg/l) to the medium was found beneficial for maximum shoot bud induction. The shoot buds developed into healthy and sturdy shoots on MS medium containing BAP and kinetin at 1.0 mg/l. Excised shoots were rooted on 1/4-strength MS medium with indole-3-acetic acid at 3.0 mg/l and 1.5% sucrose to obtain complete plants. Received: 17 June 1997 / Revision received: 11 September 1997 / Accepted: 27 September 1997     

Micropropagation of Abelmoschus esculentus L. (Moench.) for disease free plantlets through meristem culture

Protocol was established for mass in vitro propagation of okra using meristem culture. Meristems (0.3–0.5 mm in size) were isolated from shoot tips of three-week old in vitro grown seedlings. Isolated meristems were established rapidly in MS liquid medium containing 1.0 mg/l of BAP. For shoot development from primarily established meristem, semisolid MS medium having the same concentration of BAP was found to be the most effective. Rapid shoot multiplication of mericlone was achieved from node cutting cultured in 1.0 mg/l plus 0.5 mg/l GA₃, and a maximum of nine shoots were found from each node. Effective root development from the developed plantlets was successful in 1.0 mg/l IBA. More than 75% of the micropropagated mericlones plantlets were successfully acclimatised in soil up to maturity and found to be healthy.     

<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.     

Phenylacetic acid improves bud elongation and in vitro plant regeneration efficiency in Helianthus annuus L.

We have developed a highly efficient three-stage protocol for plant regeneration in sunflower (Helianthus annuus L.) from embryonal cotyledons. This protocol uses phenylacetic acid (PAA) for both shoot-bud induction and the elongation of smaller buds. The medium used for inducing bud formation from the cotyledons was modified MS medium supplemented with 3 mg/l 6-benzylaminopurine (BAP) and 0.5 mg/l PAA. Buds were elongated on MS medium supplemented either with only 0.2 mg/l gibberellic acid (GA₃) or with 0.2 mg/l GA₃ + 0.1 mg/l PAA + 0.3 mg/l BAP. The elongated shoots were then transferred onto rooting medium containing 1 mg/l PAA. The complete plantlets with well-developed roots were transferred to field conditions where they survived and set normal seeds. The induction of shoot buds from embryonal cotyledons was also observed on modified MS medium supplemented with 0.5-5 mg/l BAP in combination with 0.5-5 mg/l !-naphthaleneacetic acid (NAA). In this case, the formation of callus took place along with shoot-bud formation, which hindered further development of the latter. The presence of PAA with BAP in the primary bud induction medium promoted normal development and elongation of shoot buds.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of northern red oak (<Emphasis Type="Italic">Quercus rubra</Emphasis> L.)

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l⁻¹ casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots) to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented with 4.4 μM BA, 0.29 μM gibberellic acid (GA₃), and 500 mg l⁻¹ CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration (16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with 0.44 μM BA and 0.29 μM GA₃. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response and shoot numbers per explant were recorded on WPM supplemented with BA and GA₃, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA₃ were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20 shoots within 12 wk. Rooted plantlets were successfully acclimatized.     

A Developmental Pattern of Flowering in Colored <Emphasis Type="Italic">Zantedeschia</Emphasis> spp.: Effects of Bud Position and Gibberellin

The restricted flowering of colored cultivars ofZantedeschia is a consequence of developmental constraints imposed by apical dominance of the primary bud on secondary buds in the tuber, and by the sympodial growth of individual shoots. GA₃ enhances flowering inZantedeschia by increasing the number of flowering shoots per tuber and inflorescences per shoot. The effects of gibberellin on the pattern of flowering and on the developmental fate of differentiated inflorescences along the tuber axis and individual shoot axes were studied in GA₃ and Uniconazole-treated tubers. Inflorescence primordia and fully developed (emerged) floral stems produced during tuber storage and the plant growth period were recorded. Days to flowering, percent of flowering shoots and floral stem length decreased basipetally along the shoot and tuber axes. GA₃ prolonged the flowering period and increased both the number of flowering shoots per tuber and the differentiated inflorescences per shoot. Activated buds were GA₃ responsive regardless of meristem size or age. Uniconazole did not inhibit inflorescence differentiation but inhibited floral stem elongation. The results suggest that GA₃ has a dual action in the flowering process: induction of inflorescence differentiation and promotion of floral stem elongation. The flowering pattern could be a result of a gradient in the distribution of endogenous factors involved in inflorescence differentialtion (possibly GAs) and in floral stem growth. This gradient along the tuber and shoot axes is probably controlled by apical dominance of the primary bud. Online publication: 7 April 2005     

A rapid in vitro propagation of red sanders (<Emphasis Type="Italic">Pterocarpus santalinus</Emphasis> L.) using shoot tip explants

An efficient protocol has been developed for the in vitro propagation of Pterocarpus santalinus L. using shoot tip explants which is a valuable woody medicinal plant. Various parts of this plant are pharmaceutically used for the treatment of different diseases. Multiple shoots were induced from shoot tip explants derived from 20 days old in vivo germinated seedlings on 1:1 ratio of sand and soil after treating with gibberellic acid (GA₃). The highest frequency for shoot regeneration (83.3%) with maximum number of shoot buds (11) per explant was obtained on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l of 6-benzylaminopurine (BAP) along with 0.1 mg/l of thidiazuron (TDZ) after 45 days of culture. A proliferating shoot culture was established by repeatedly subculturing the original shoot tip explants on fresh medium after each harvest of the newly formed shoots. Sixty percent of the shoots produced roots were transferred to rooting medium containing MS salts and 0.1 mg/l indole-3-butyric acid (IBA) after 30 days. About 73.33% of the in vitro raised plantlets were established successfully in earthen pots. Random amplified polymorphic DNA (RAPD)-based DNA fingerprinting profiles were generated for the first time using shoot tip explants of this species and confirmed that there was no genetic variability. This protocol might be helpful for the mass multiplication of P. santalinus in the future.     

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 %.     

In vitro microrhizome production in Zingiber officinale Rosc.

Microrhizomes of Zingiber officinale were successfully produced from tissue culture derived shoots by transferring them to liquid MS medium supplemented with 1 mg/l BAP, 2 mg/l calcium pantothenate, 0.2 mg/l GA₃ and 0.05 mg/l NAA for shoot proliferation. After 4 weeks of incubation, the medium was replaced with microrhizome induction medium, consisting of MS salts supplemented with 8 mg/l BAP and 75 g/l sucrose. Microrhizome formation started after 20 d of incubation in stationary cultures at 25+1 ° in the dark. Microrhizomes with 1–4 buds and weighing 73.8 to 459 mg each were harvested after 50–60 d. After storage for 2 months in moist sand at room temperature, 80% of the microrhizomes sprouted producing roots and shoots.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

The role of GA,IAA and BAP in the regulation of <Emphasis Type="Italic">in vitro</Emphasis> shoot growth and microtuberization in potato

The effect of auxin, GA and BAP on potato shoot growth and tuberization was investigated under in vitro condition. The shoot length of potato explants increased with the increasing of concentrations (0.5 – 10 mg dm⁻³) of IAA treatment especially with the addition of GA₃ (0.5 mg dm⁻³), but was inhibited by BAP (5 mg dm⁻³). The root number and root fresh weight of potato explants increased with the increasing of IAA levels either in the presence of GA₃ (treatment IAA+GA) or not (IAA alone). However, no root was observed in the treatment IAA+BAP, instead there were brown swollen calli formed around the basal cut surface of the explants. The addition of GA₃ remarkably increased the fresh weight and diameter of calli. Microtubers were formed in the treatments of IAA+BAP and IAA + GA + BAP but not observed in the treatments of IAA alone or IAA + GA. IAA of higher concentrations (2.5 – 10 mg dm⁻³) was helpful to form sessile tubers. With the increasing of IAA levels, the fresh weight and diameter of microtubers increased progressively. At 10 mg/L IAA, the fresh weight and diameter of microtubers in the treatment of IAA + GA + BAP were 409.6 % and 184.4 % of that in the treatment of IAA + BAP respectively, indicating the interaction effect of GA and IAA in potato microtuberization.     

Efficient Plant Regeneration System From Leaf Discs of Zonal (Pelargonium x hortorum) and two scented (P. capitatum and P. graveolens) Geraniums

An efficient and rapid plant regeneration system was established for zonal and scented geraniums using leaf discs as explants. Several explants, medium and culture conditions were studied to optimize shoot induction. Leaf discs taken from 4–5 weeks old in vitro grown plants, whatever the genotype, were more effective for shoot regeneration than those taken from greenhouse grown plants. Darkness proved to be a stimulating factor for shoot regeneration and the combination between NAA and two cytokinins gave the best results. Direct shoot regeneration (100%) was obtained from leaf discs of P. capitatum on half-strength MS medium supplemented with 0.5 mg l⁻¹ NAA in combination with 1 mg l⁻¹ of BAP and zeatin in darkness (11.4 shoots per explant). In the same medium and culture conditions, all P. graveolens leaf discs also exhibited direct shoot regeneration (7.3 shoots per explant). For P. x hortorum, 100% of leaf discs underwent shoot regeneration on a MS medium supplemented with 0.2 mg l⁻¹ NAA in combination with 0.5 mg l⁻¹ of BAP and zeatin in darkness (8.8 shoots per explant) or under low light conditions with 0.2 mg l⁻¹ NAA and 1 mg l⁻¹ of BAP and zeatin (7.5 shoots per explant). For this species, the best results for shoot elongation were obtained on half-strength MS medium gelled with Phytagel 0.3% (v/v). Whatever the genotype, all shoots rooted readily when transferred to diluted MS medium (MS/2) containing 1 mg l⁻¹ IAA. Acclimatized plants grew normally and flowered in greenhouse conditions. Flow cytometry analysis made on leaves of acclimatized plants revealed that all the scented geranium plants are similar to mother plants while 71% of P. x hortorum plants which showed strong growth were tetraploid.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

High efficiency plant regeneration and genetic fidelity of regenerants by SCoT and ISSR markers in chickpea (Cicer arietinum L.)

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l^?1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l^?1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l^?1 BAP along with 0.05 mg l^?1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l^?1), GA₃ (1.0 mg l^?1) and BAP (1.0 mg l^?1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l^?1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation.