Maximum Number Of Multiple Shoots Research Articles

Cost Effective Methods of in Vitro Multiple Shoot Production in Two Commercial Diploid Bananas of Kerala

Cost-effective methods for in vitro propagation of two commercial diploid bananas of Kerala, namely Musa (AB) ‘ Njalipoovan’ and Musa (AA) ‘ Nivedyakadali’ were carried out to reduce unit production cost. Sword sucker, peeper sucker and eye bud explants were found to be equally effective concerning the number of axillary shoots from a single explant in both cultivars. Sucrose 3.0 per cent and table sugar 2.0 per cent produced the maximum number of multiple shoots in Njalipoovan. In the case of Nivedyakadali, table sugar at 2.0 per cent recorded the maximum number of multiple shoots. Half strength of vitamins in full MS medium observed more multiple shoots in cv. Njalipoovan. Half a tablet of vitamin B complex (2.0mgl-1) in the medium resulted in more shoots in the case of cv. Nivedyakadali. Agar 0.7 per cent recorded a significantly higher number of multiple shoots per culture in both cultivars. The treatments involving different types of paper support (filter paper, brown paper and ordinary paper) in liquid media were inferior to agar 0.7 per cent treatment.

IN-VITRO REGENERATION OF MULTIPLE SHOOTS FROM APICAL MERISTEMS OF COWPEA (Vigna unguiculata L. (WALP) USING DIFFERENT GENOTYPES (LOCAL AND IMPROVED) AND MEDIA COMPOSITIONS

Organogenesis in-vitro was evaluated in explants derived from shoot apices of four cowpea varieties cultured on Murashige and Skoog (MS) basal medium. To evaluate the response of the cowpea genotypes to in vitro regeneration, seedlings were drived from zygotic embryos excised from surface sterilized seeds after two weeks of culture on MS basal medium + vitamins supplement with 30% sucrose. To regenerate multiple shoots, apices of the in vitro seedlings with average length of 1cm were excised and cultured on MS supplemented with 0.5, 1.0 or 1.5mg/L 6-Benzylamino purine(BAP) with or without 0.1mg/L α-naphthaleneacetic acid (NAA) for six weeks. The highest shoot number was recorded with IT99k-573-2-1 and Danilla and shoot length was highest in Kanannado, IT04k-33-2-1and Danilla indicating that Danilla responded well to in vitro regeneration. To optimise the production of multiple shoots the effects of different concentratios (0.5, 1.0 and 1.5mg/L) of 6-benzyaminopurine (BAP) with or without 0.1mg/L α-naphthaleneacetic acid on the number of responding explant, number of multiple shoot produced and length of the shoots were tested. after six weeks of culture, the percentage of responding shoot apices was highest on media supplemented with 1.5mg/L BAP with and without 0.1mg/L NAA.The number of responding apices decreasing with decrease in the concentration of BAP. Maximum number of multiple shoots and the highest mean shoot length were also obtained when MS was supplemented with 1.5mg/L BAP with and without 0.1mg/L NAA.

In vitro propagation and DNA barcoding of the rare medicinal plant Aerva Lanata (L.) Juss. ex Schult grown in Gebel Elba

Aerva Lanata (L.) Juss. ex Schult is an important medicinal multipurpose plant that belongs to the family Amaranthaceae. An efficient protocol for In vitro propagation of A. Lanata was established. Nodal segment explants collected from mature shrubs from the Gebel Elba, South Egypt. Explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyl adenine (BA) (2.22, 4.44, 8.90 and 17.80 M) and Kinetin (Kin) (2.32, 4.60, 9.30, and 18.6M) individually for shoot induction. The highest shoot induction percentage (93.33%) with maximum number for shoots (4.07 shoots per explant) was noticed on MS medium supplemented with 4.44 M BA. The maximum number of multiple shoots per explants (9.13) was obtained in the combination BA (4.44 M) and NAA (2.14 M). For root induction, the highest rooting percentage (80%) with mean root number (4.33 roots per shoot) and length (3.83 cm) of roots were noticed on half- strength MS medium augmented with 4.90 M IBA. Rooted plantlets were transferred into plastic pots containing sand, peat moss and garden soil mixture at equal volumes, and successfully acclimatized in the greenhouse with a survival rate of 73.3%. Plant identification is a crucial aspect to understand and conserve plant diversity from extinction. DNA barcode analysis of A. Lanata was carried out using. The present in vitro propagation protocol would facilitate an alternative method for rapid and large-scal production of this important medicinal plant. We therefore, used the rbcL gene to identify A. lanata. Maximum likelihood tree analysis was performed to evaluate the discriminatory power of the rbcL gene. Our findings showed that using rbcL gene sequences enabled identifying the majority of the samples (90%) to the genus and species level.

In vitro micro-propagation of Typhonium flagelliforme (Lodd.) Blume and its genetic fidelity using ISSR and RAPD markers

An araceous plant, Typhonium flagelliforme is in its extinct stage and require several ways to conserve its family. In the present study, an efficient In vitro protocol is established using apical meristem as an explant on basal Murashige and Skoog media supplemented with different concentrations of plant growth regulators. As an outcome, maximum number of multiple shoots (10.7±0.67) were generated on the media supplemented with 2.0 mg/L 6 - benzylaminopurine (BAP) and 0.5 mg/L α-napthalene acetic acid (NAA). The regenerated shoots were further sub-cultured on the fresh media containing 0.5mg/L BAP for elongation and proliferation. Media supplemented with 1.0 mg/L Indole-3-butyric acid (IBA) and 2% sucrose enhanced rooting in the proliferated shoots. In the process, an average of 90% plantlets survived, hardened and transferred to the field conditions. Further, genetic fidelity was exhibited among the tissue culture raised plants with the markers ISSR and RAPD. Thus, the modified tissue culture protocol and its standardization would be used for large-scale propagation and conservation of this medicinal plant.

In vitro micropropagation and genetic fidelity studies using SCoT and ISSR primers in Annona reticulata L.: an important medicinal plant

An efficient and reproducible in vitro plant regeneration protocol has been developed for Annona reticulata L., a medicinally important plant of family Annonaceae. This is the first report of an efficient in vitro micropropagation and genetic fidelity evaluation of Annona reticulata using nodal segments as explants. Nodal explants were cultured on Murashige and Skoog (MS) medium augmented with various concentrations of cytokinins alone and in combination with auxins. MS medium supplemented with 2.5 mg/l 6-benzylaminopurine (BAP) and 0.3 mg/l indole-3-butyricacid (IBA) combination has proven the best for optimum regeneration response (87.4%) and a maximum number of multiple shoots (6.33 ± 0.37). Rhyzogenesis occurred on half-strength MS medium supplemented with 4 mg/l IBA, resulted in the highest percentage of rooting (73%) and a maximum number of roots (4.65 ± 0.23) along with maximum root length (1.76 ± 0.08 cm). The well developed regenerates were shifted to the mixture of soil, sand and organic manure (2:1:1) in small plastic cups and acclimatized successfully. Genetic fidelity of the discussed etiquette was validated using two types of DNA fingerprinting techniques i.e. start codon targeted (SCoT), and inter simple sequence repeat (ISSR) analyses. Among the ten SCoT (SC) and ISSR primers used, an excellent amplification with scorable DNA bands was produced by SC9 and ISSR4 primers. The results showed that the regenerated plantlets are monomorphic and true-to-type with mother plant. This study provides important information about selection of suitable regeneration medium to improve multiple shoot induction and rooting of Annona reticulata. It can be used for commercial cultivation and for further genetic redevelopment investigation studies, also applied for large scale propagation of elite genotypes.

Meta-topolin and β-cyclodextrin enhance multiple shoot and root production in black gram Vigna mungo (L.) Hepper

The recalcitrant nature of black gram is the major constraint of in vitro regeneration and agrobacterium- mediated genetic transformation, to overcome this, a productive shoot regeneration protocol has been achieved in black gram cultivar T9 using 7-day old cotyledonary node explants excised from in vitro- raised black gram seedlings using meta-topolin. An aromatic cytokinin,meta-topolin along with BA (1.5+0.5 mg/L) in 0.50 strength MS medium with 1.5% (w/v) sucrose exhibited a maximum number of multiple shoots (32.0±0.37 shoots/explant) at the end of 6 weeks of culture. The shoots were elongated in (6.40±0.50 cm/shoot) in MS medium supplemented with GA3 (2.0 mg/L). A maximum number of roots (9.60±0.50/shoot) and root length (11.20±0.73 cm/shoot) were obtained in combination with β-cyclodextrin(a cyclic oligosaccharide;1.5 mg/L) and IBA (1.5 mg/L). The rooted plantlets were hardened and acclimatized with least mortality rate of 2% in pot mixture consisting red soil:sand:farm yard manure (FYM) (2:1:1) and grown in green house with 85% relative humidity. Ploidy levels were analyzed using flow cytometry which confessed the chromosomal stability in invitro raised plants similar to parent plants. This protocol may be useful for producing transgenic black gram with desirable agro-traits in Indian cultivars.

In Vitro Direct Regeneration through Cotyledon Culture in Pigeon Pea [Cajanus Cajan (L.) Millsp.] and Evaluation of Genetic Fidelity using RAPD Markers

In Vitro Direct Regeneration through Cotyledon Culture in Pigeon Pea [Cajanus Cajan (L.) Millsp.] and Evaluation of Genetic Fidelity using RAPD Markers An efficient and direct in vitro regeneration of Cajanus cajan (red gram) cv ICP 26 from cotyledon explants was established. Cotyledons of 7-8 day old in vitro grown seedlings were cultured on MS/B5 medium augmented with different concentrations (0.5-5.0 mg/L) of 6-benzylaminopurine (BAP) / Kinetin (KIN). Among these concentrations, 2.0 mg/L BAP was found to be efficient in inducing maximum number of multiple shoots with 80% of response. 80% of the elongated shoots were successfully rooted on MS medium fortified with 1.0 mg/L indole-3-butyric acid (IBA). These in vitro rooted plantlets were successfully established in soil where 80- 90% of them developed into normal and fertile plants. 20 RAPD primers were used to assess the genetic fidelity of randomly selected regenerated plantlets along with the donar plant. Out of 20 RAPD primers screened only 7 primers produced clear, distinct and scorable bands with an average of 2.5 bands per primer. All banding profiles from regenerated plantlets were monomorphic and similar to those of the mother plant. The amplification pattern confirmed the genetic uniformity of the regenerated plantlets. Thus, this reproducible regeneration protocol can be used for Agrobacterium tumefaciens mediated genetic transformation in pigeon pea....

Enhancing propagation efficiency of banana cv. MONTHAN (ABB) through micropropagation

An investigation was carried out at Plant Tissue Culture Laboratory of Horticultural College and Research Institute, Coimbatore during 2012-2013. Shoot tip of banana cv. MONTHAN were cultured in MS medium fortified with different growth regulators (BAP @ 4, 5 and 6 mg l -1 ) either alone or in combination with NAA (0.5 mg l -1 ) and kinetin (2 mg l -1 ) to study the influence of growth regulators on multiplication rate. The data on number of multiple shoots, days for multiple shoot induction and micro shoot length were recorded. MS+BAP 6 mg l -1 + kinetin 2 mg l -1 recorded maximum number of multiple shoots (5.07). MS + BAP 6 mg l -1 recorded least days for multiple shoot induction (27.06 days) and highest micro shoot length (6.73cm). Further to enhance multiplication best growth regulator combination (MS + BAP 6 mg l -1 + Kinetin 2 mg l -1 ) along with different concentration of coconut water (5, 10 and 15%) and casein hydrolysate (100, 200 and 300 mg l -1 ) were supplemented. MS + BAP 6 mg l -1 + kinetin 2 mg l -1 +coconut water 5 per cent recorded the highest number of multiple shoots at all the intervals of subculture (5.13). The best growth regulator combination (MS+BAP 6 mg l -1 + kinetin 2 mg l -1 ) along with B5 vitamins in specific to different thiamine concentration (5, 10 and 15 mg l -1 ) for further multiplication were supplemented and B 5 vitamins with thiamine combinations did not increase the multiplication rate. The degree of efficiency of shooting was found to be dependent on type of hormone.

Micropropagation of an important medicinal herb Eclipta alba (L.) Hassk.

Nodal segments from naturally grown Eclipta alba (L.) Hassk.were used as explants for organogenesis. Multiple shoots were obtained from the explants cultured on MS medium supplemented with various concentrations of BAP and Kn alone or in combination with NAA and IAA. Maximum number of multiple shoots (18.40±0.67) were induced on MS medium supplemented with 1.0 mg/l BAP and 0.1mg/NAA. In vitro raised shoots were cultured onto half and full strength MS medium supplemented with different concentration of IBA, IAA and NAA. The best root induction medium was found to be half strength MS containing 0.1 mg/l IBA where 96% shoots rooted. Regenerated plantlets grew normally without showing any morphological variation and flowered after 45 days of transplantation.Jahangirnagar University J. Biol. Sci. 4(1): 61-69, 2015 (June)

Establishment of in vitro regeneration protocol for Adhatoda vasica Nees

An in vitro regeneration protocol of Adhatoda vasica has been developed using excised nodal segments and juvenile leaves for multiple shoots regeneration directly or through callus induction. Explants were cultured on MS medium with different concentrations of IAA, NAA, BAP, GA3 and Kn singly or in combinations. MS medium supplemented with BAP (10.0 mg/l) was found best for multiple shoot formation, in which 93.33% explants produced multiple shoots. After two months, maximum number of multiple shoots were 10.6 ± 1.82, highest length of plantlets was 5.2 ± 2.20 cm. 100% calli formation were observed on MS medium supplemented with IAA (0.05 mg/l) + NAA (0.05 mg/l) + BAP (1.0 mg/l). Callus initiation started after 14 days and gave light green colored callus. Best callus mediated shoot regeneration was found on MS+10.0 mg/l BAP medium. Root induction of in vitro raised shoots was best on ½ MS + IBA (1.0 mg/l). Well rooted plantlets were transferred to plastic pots containing garden soil and compost in a ratio of 2:1 for hardening. The ultimate survival rate under natural condition was about 80%.Bangladesh J. Sci. Ind. Res. 51(1), 75-80, 2016

Role of Silver Nitrate on <i>in Vitro</i> Flowering and Shoot Regeneration of <i>Solanum nigrum</i> (L.)—An Important Multipurpose Medicinal Plant

Silver nitrate induced MS media enhanced multiple shoot regeneration and in vitro flowering from axillary bud and leaf explants of Solanum nigrum (L.) an important antiulcer medicinal plant. Healthy axillary bud and leaf explants were inoculated on MS medium supplemented with BAP/Kn (1.0 - 2.0 mg/l) in combination with IAA/NAA (0.5 mg/l) and AgNO3 (0.1 - 1.0 mg/l). The explants were responded effectively and good regeneration frequencies were observed in all the combinations of silver nitrate tested when compared to control. Maximum number of multiple shoots (34.3) was found in leaf explants cultured on MS media supplemented with BAP (2.0 mg/l), NAA (0.5 mg/l) and AgNO3 (0.4 mg/l). These regenerated shoots were sub-cultured on to the flowering media. Maximum number of in vitro flowers (12) was obtained from axillary bud explants in BAP (2.0 mg/l), Kn (1.0 mg/l), IAA (0.5 mg/l) and AgNO3 (6.0 mg/l) supplemented media when compared to leaf (4) and control. All the in vitro raised shoots were transferred to rooting medium supplemented with NAA, IBA (1.0 - 2.0 mg/l) and AgNO3 (0.1 - 0.6 mg/l). The best rooting response (24.6) was observed in 2.0 mg/l IBA + 0.4 mg/l AgNO3. The well rooted plantlets were transferred to polybags containing soil + vermiculate in 1:1 ratio for hardening and finally the hardened plantlets were transferred to field conditions.

Callus Induction and Organogenesis in Sugarcane (<i>Saccharum officinarum</i> L.) var 93v297

An efficient protocol for induction of callus and regeneration of a sugar cane var 93v297 has been developed and reported here. Callus induction from immature young leaf explants derived from 2-3-month-old plants was achieved on Murashige and Skoog’s (MS) medium supplemented with different auxins viz, 2,4-D, NAA and IAA. Among different auxins, 2, 4-D at 3.5mg/l + 0.5mg/l BAP was found favourable in inducing callus. Addition of coconut milk and BAP further enhanced the growth of callus maximum being on MS medium supplemented with 0.5mg/l BAP (3602.33±0.88mg). Calli were further evaluated for regeneration. MS medium supplemented with 1.0 mg/l BAP was found suitable where 100% calli regenerated with maximum number of multiple shoots per callus mass (41.40±0.89). Highest number of root emergence (28.33±1.16) and maximum root length (3.40±0.67cm) was achieved on MS medium supplemented with 3mgl/l NAA. The in vitro grown plants were transferred to polycups containing a mixture of sterilized sand, soil and cocopeet (1:1:1) for hardening. The hardened plants were transferred to green-house conditions where they survived with 90% frequency.

Callus Induction and Organogenesis in Sugarcane (<i>Saccharum officinarum</i> L.) var 93v297

An efficient protocol for induction of callus and regeneration of a sugar cane var 93v297 has been developed and reported here. Callus induction from immature young leaf explants derived from 2-3-month-old plants was achieved on Murashige and Skoog’s (MS) medium supplemented with different auxins viz, 2,4-D, NAA and IAA. Among different auxins, 2, 4-D at 3.5mg/l + 0.5mg/l BAP was found favourable in inducing callus. Addition of coconut milk and BAP further enhanced the growth of callus maximum being on MS medium supplemented with 0.5mg/l BAP (3602.33±0.88mg). Calli were further evaluated for regeneration. MS medium supplemented with 1.0 mg/l BAP was found suitable where 100% calli regenerated with maximum number of multiple shoots per callus mass (41.40±0.89). Highest number of root emergence (28.33±1.16) and maximum root length (3.40±0.67cm) was achieved on MS medium supplemented with 3mgl/l NAA. The in vitro grown plants were transferred to polycups containing a mixture of sterilized sand, soil and cocopeet (1:1:1) for hardening. The hardened plants were transferred to green-house conditions where they survived with 90% frequency.

Micropropagation and Attempts to Develop RAPD Molecular Markers for Salt Tolerant Cultivars of Pearl Millet

Differences were observed among four salt tolerant cultivars of pearl millet to in vitro culture media. Among the four cultivars, Cv IP 6098 is the most salt sensitive had maximum number of multiple shoots (15)/ seed, and the more salt tolerant cvs IP 6106 and IP 22269 developed 3-6 shoots/seed while cv IP 19612 the seeds remained single shooted in MS+3.0mg/L media up to 7 months. The multiple shoots 20% of cv IP 6098, flowered in 5 months with the reduction in hormone concentration (MS+1.0 mg/L BAP) whereas, the other three cultivars did not flower under the tested conditions.
 The present study was carried to detect genetic differences among the four cultivars of pearl millet at molecular level through RAPD PCR markers. RAPD analysis was carried with 12 primers, each 10-mer, only 7 primers were amplified showed polymorphism for the four cultivars. Primer OPM 16 revealed a single unique band of approx. 485bp in the genotype IP 22269 and three polymorphic bands in the genotype IP 6106. The results were repeatable, and further studies in this direction may enable to link the single band in the genotype IP 22269 to a morphological trait or physiological condition like salt tolerance.

L-Dopa production and antioxidant activity in Hybanthus enneaspermus (L.) F. Muell regeneration.

Hybanthus enneaspermus is an ethanobotanical plant extensively used in Indian traditional medicine. Quick and efficient in vitro mass propagation of this plant species was established for commercial utilization from leaf and node explants using various concentrations and combinations of plant growth regulators and polyamines. The maximum number of multiple shoots per leaf explant (40 shoots) was achieved on MS medium supplemented with 20mg/l spermidine in combination with 4mg/l BA+1.5mg/l IAA after 8weeks of culture. The elongated shoots were rooted (16 roots/shoot) on MS medium with the best concentration of IBA (1.5mg/l) and in combination with 20mg/l putrescine after 5weeks of culture. The plants were successfully acclimatized (98%) in the sand: soil: vermiculite mixture (1:1:1 v/v/v) in the greenhouse. An increased antioxidant activity was recorded in vitroregenerated shoots when compared to in vitro-induced roots. L-Dopa content was recorded higher in leaves (8.31mg/g DW) followed by stem (6.22mg/g DW) and root (3.22mg/g DW) of leaf-derived plants than the field-grown parent plant after 5weeks. By adopting this protocol, the regenerated-plants could be used for drug production and pharmacology work with as an alternative to field-grown plants.

In vitro plantlet regeneration from cotyledon segments of Capsicum chinense Jacq. cv. Naga King Chili, and determination of capsaicin content in fruits of in vitro propagated plants by High Performance Liquid Chromatography

An in vitro plantlet regeneration system has been developed from cotyledon segments of Capsicum chinense Jacq. cv. Naga King Chili, a very pungent chili species of India. Rosette-like structures (RLS) were induced on the explants which on transfer to medium containing indole-3-acetic acid (IAA) resulted in multiple shoots in course of time. Agar-based Murashige and Skoog (MS) medium fortified with 18.16μM thidiazuron (TDZ) was found to be the most suitable medium for RLS induction and shoot formation. Maximum number of multiple shoots (9.5±0.39) and roots (8.6±0.50), and root length (2.4±0.02cm) were obtained from rosette-like structures in medium containing 5.70μM IAA. Combined effect of putrescine (Put) (5.6μM) and TDZ (4.54μM) mediated direct multiple shoot (5.8±0.44) induction. The in vitro rooted plantlets were transferred to glass house for hardening and acclimatization, wherein 90% survival was recorded. The hardened plantlets, thus developed were established in soil and bore normal fruits after 4 months of transfer. The capsaicin content in matured fruits of in vitro propagated plants was found to be 0.05236g/gdwt (837,760 Scoville Heat Units, SHU), where as in vivo raised plantlets yielded 0.0545g/gdwt (872,000 SHU) respectively.

In vitro clonal multiplication of Aegle marmelos (L.) Corr. through cotylodonary node culture

Clonal multiplication of Aegle marmelos was achieved through in vitro culture. Cotyledonary nodes from one month old in-vitro grown seedlings of Aegle marmelos were cultured on MS medium supplemented with BAP, Kn, and IBA either alone or in combination. The highest regenerative response was observed on medium containing 2.5 mg/L BAP and 0.5 mg/L Kn from node and shoot tip within 8-10 days. When regenerated shoots were subcultured on MS medium supplemented with 0.2 mg/L BAP and 0.02 mg/L Kn, maximum number of multiple shoots were observed after third phase of subculture. Poor response was found using MS medium supplemented with Kn only. In vitro induced shoots were transferred into root induction medium consisting of half-strength MS supplemented with auxins, IAA, IBA or NAA. Rooting was best in medium supplemented with 1.0 mg/L IBA. Rooted plantlets were acclimatized and transferred to the soil with 96% survival rate. DOI: http://dx.doi.org/10.3329/bjsir.v48i1.15408 Bangladesh J. Sci. Ind. Res. 48(1), 13-18, 2013

Lipid peroxidation, H<sub>2</sub>O<sub>2</sub> content, and antioxidants during acclimatization of Abrus precatorius to ex vitro conditions

An efficient, rapid, and reproducible plant regeneration protocol was successfully developed for Abrus precatorius L. using mature nodal explants excised from a 5-year-old field grown plant. The highest shoot regeneration frequency (87 %) with maximum number of multiple shoots (15.0) and shoot length (4.8 cm) were recorded on Murashige and Skoog (MS) medium amended with 2.5 μM thidiazuron, 120 mg dm−3 polyvinylpyrrolidone, and 0.5 μM α-naphthalene acetic acid. The best treatment for maximum root (4.0) induction was half strength MS medium supplemented with 1.5 μM indole-3-butyric acid. The in vitro plantlets with well-developed shoots and roots were successfully transferred into plastic cups with Soilrite and acclimatized in a culture room under photon flux density (PFD) of 150 μmol m−2 s−1, thereafter transferred to a greenhouse with PFD of 300 μmol m−2 s−1, and finally to a field with 70 % survival rate. During the acclimatization period (0–49 d), leaf chlorophyll and carotenoid content increased whereas malondialdehyde and H2O2 content decreased probably due to increasing activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase). Our work suggests that micropropagated plants developed an antioxidant enzymatic protective system to avoid oxidative stress during establishment under ex vitro environment.

Efficient in vitro Regeneration of a Medicinal Plant Harsinghar (Nyctanthes arbor-tristis L.)

In vitro propagation of Nyctanthes arbor-tristis L. has been successfully established from axillary bud explants on MS. Maximum number of multiple shoots was obtained on MS containing BAP (22.2 ?M). Half strength of MS (2% sucrose) supplemented with NAA (10.74 ?M) provided the maximum frequency of root initiation. The plantlets were successfully hardened. DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14202 Plant Tissue Cult. & Biotech. 22(2): 137-142, 2012 (December)

High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex

Cotton (Gossypium hirsutum L.) is one of the most commercially important fiber crops in the world. Compared with other crops, cotton represents a recalcitrant species for regeneration protocols. The development of efficient and rapid regeneration protocol for elite Indian cotton variety could help improve the quality characteristics and biotic or abiotic stress tolerance. Here we report a novel regeneration protocol in Indian cotton cultivar Narashima. The maximum number of multiple shoots obtained was 16 per explants, performance which has never been achieved in any prior reports. The embryo apex explants were isolated from 2 d old in vitro growing seedlings. Explants were cultured on MS medium containing different plant growth regulator combinations in order to induce multiple shoots. Among the tested combinations, the 2 mg/l of 6-benzylaminopurine (BAP) and 2 mg/l kinetin (KIN) proved to be most suited for achieving the maximum number of multiple shoots. The elongation of multiple shoots was obtained in media supplemented with gibberellic acid (GA3). The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. This method avoids callus tissue, the stage of regeneration which may lead to somaclonal variation. The important feature of the presented method is shortening of regeneration time, as well as the induction of a high number of multiple shoots per explants. The present protocol may provide an efficient and rapid regeneration tool for obtaining more stable transformants from embryo apex explants of Indian cotton cultivar Narashima.