Multiple Shoot Regeneration Research Articles

English

Myrica rubra is an important horticultural, forestry and ornamental tree. Its micropropagation was achieved by using shoot tip and nodal explants from two commercial cultivars “Biji and Dongkui” on BW medium supplemented with Thidiazuron (TDZ) and/or 2,4-Dichlorophenoxyacetic acid (2,4-D). TDZ induced multiple shoots but stunted growth. Maximum number of shoots (5.75) was recorded for both cultivars with 0.6 mg L-1TDZ concentration. Shoot length was significantly improved with optimum concentration of TDZ (0.6 mg L-1) and with different  concentrations of 2,4-D. BW medium was found as suitable medium for micropropagation of both the cultivars of M. rubra with maximum number and longer shoots. WPM and MS medium ranked second in multiple shoot regeneration of both cultivars. Single microshoot cultured on plant growth regulator free medium for 3 weeks initiated roots on the medium containing  Indole butyric acid (IBA) only with reduced (¼ and ½) medium strength. Roots were induced with a high frequency of 95% in Biji and 87% in Dongkui with 0.3 mg L-1 IBA, respectively. No callus was observed on shoots in rooting media. All the concentrations of indole-3-acetic acid (IAA) either on reduced medium or full strength did not initiate any roots. Rooted plants were successfully acclimatized.   Key words: Myrica rubra, Chinese bay berry, micropropagation, plant growth regulators.

An improved method of Agrobacterium tumefaciens-mediated genetic transformation system of melon (Cucumis melo L.)

A reliable Agrobacterium-mediated transformation and shoot regeneration protocol was developed for breeding lines of commercially important melon. Genetic manipulation has been considered a feasible approach for melon improvement; however, melon is considered a crop species difficult to manipulate. Here we proposed meristematic cells from mature embryos as target for gene transfer by Agrobacterium. In vitro meristems proliferation and multiple shoots regeneration were evaluated by sowing melon mature seeds on MS with 1.0 mg/L benzyladenine (BA), and 0.05 mg/L indole acetic acid (IAA) were used for shoot regeneration. The highest number of regenerated shoots was obtained from half mature seeds. A DNA fragment corresponding to selection marker nptII was amplified from genomic DNA extracted from leaves of regenerated plant on hormone free MS medium with 75 mg/L kanamycin, suggesting their transgenic nature. Southern hybridization of transgenic lines revealed random insertion of the transgene in host genome, with insert numbers differing among transformants anthesis, suggesting that ethylene is important for sex determination. Field studies showed that CmACS-7 melons had earlier mature bisexual flowers, increased femaleness as measured by earlier and bisexual buds, and increased number of fruit set on closely spaced nodes on the main stem. Transformation efficiencies of cultivar CM-23 with EHA105 (pBI121-cm) were 4 %, demonstrating that melon meristematic cells are an useful target for genetic manipulation by agroinfection.

In vitroPlant Regeneration in Brinjal (Solanum melongenaL.) Using Cotyledon, Hypocotyl and Root Explants

The present investigation was undertaken for developing a high frequency regeneration system, as an essential pre-requisite for the genetic transformation in brinjal. Regeneration of multiple shoots via callus induction and organogenesis was achieved using cotyledons, hypocotyls and roots as explants at various concentrations and combinations of growth regulators. Hundred percent callus induction was observed in cotyledon and hypocotyl explants when cultured on medium supplemented with BAP or Zeatin. Earliest callus induction was observed in cotyledon explants (8.03 days). The MSB5 media supplemented with 2 mg/l zeatin + 0.1 mg/l IBA found to be the best medium with regards to number of days requires for callus induction and shoot induction, number of shoot buds formed per explant and regeneration frequency. Among explants used cotyledons found to be give best response to shoot induction and number of shoot bud formation (87.50% and 24) and followed by hypocotyls (83.33% and 22) and root explant (69.17% and 12). In vitro rooting (53.33%) was obtained within 13 days on MSB5 media supplemented with 1mg/l IBA. The in vitro rooted plantlets were successfully established in polycabonated polyhouse with 92% survival rate.

Regeneration of multiple shoots from petiole callus of Viola serpens Wall

Experiments were conducted to develop methodology for in vitro propagation and rapid multiplication of Viola serpens Wall. using petiole explant. The MS medium supplemented with 2, 4-D (6.78 μm) was found most suitable for callus induction in petiole explant. The best growth response and higher rate of shoot regeneration from petiole callus was observed on MS medium containing BAP (11.10 μm) as the average number of shoots could be increased to 36.4 on fourth successive subculturing. Higher rooting response with larger number of roots were observed in shoots inoculated on the half-strength MS medium supplemented with IBA (19.68 μm).

Multiple shoot regeneration of plumular apices of chickpea

Chickpea (Cicer arietinum L.) is an important grain legume used almost all over the world. Development of new repeatable and reliable tissue culture protocols is very important for improvement of breeding and genetic transformation studies. It is a highly recalcitrant plant and there is an urgent need to develop a regeneration protocol that can ensure easy multiple and qualitative superior shoots that could be rooted and yield fertile plants. Unconditioned plumular apices and plumular apices preconditioned with 10 mg L^{-1} benzylaminopurine (BA) for 10 days were cultured on Murashige and Skoog (MS) medium supplemented with 0.25-2.00 mg L^{-1} BA with or without 0.25 mg L^{-1} naphthalene acetic acid (NAA). Comparing the 2 culture conditions, the preconditioned explants had 2-fold to 5-fold more regeneration and shoots per explant compared to the unconditioned plumular apices. The presence of NAA in the culture medium positively increased the number of shoots per preconditioned explant at the lower concentrations of BA. In contrast, NAA inhibited the number of shoots per explants and the mean shoot length of the unconditioned explants at all concentrations of BA. An increased sucrose concentration of 45 and 60 g L^{-1} (R1) with 1.00 mg L^{-1} indole-3 butyric acid resulted in 50% rooting with multiple and earlier hardening of shoots. Subculturing of multiple shoots on MS medium containing 45-60 g L^{-1} sucrose (R2) enhanced the rooting frequency by 60%-100%. The rooted plantlets were successfully acclimatized.

In- vitro regeneration of an important medicinal plant Centella asiatica L. Urban

An in vitro multiple shoot regeneration protocol has been developed for Centella asiatica L. Urban from the nodal and shoot tips explants using MS medium with different concentrations and combinations of growth regulators. Multiple shoot regeneration was observed from both the explants on MS containing either BAP alone or in combination with IAA, NAA and Kn. Of the two explants, nodal segment showed comparatively better response towards multiple shoot regeneration. Maximum multiple shoots were found on MS supplemented with 1.0 mg/L BAP and 0.4 mg/L NAA. For root induction, well-developed shoots were excised and cultured on both MS and half strength of MS with various concentrations of IBA, IAA and NAA. Profuse healthy rooting was obtained on MS medium containing 0.2 mg/L IBA. The well rooted plantlets were successfully transplanted to the garden soil and their survival rate under natural condition was 90-95 %. DOI: http://dx.doi.org/10.3329/bjsir.v47i3.13058 Bangladesh J. Sci. Ind. Res. 47(3), 269-272, 2012

Effect of thidiazuron (TDZ) on in vitro regeneration of blackgram (Vigna mungo L.) embryonic axes

Regeneration has been achieved in blackgram (Vigna mungo) using thidiazuron (TDZ) in the culture medium. The explanted cotyledon with wounded embryonic axes produced the highest number (9.75–10.45) of healthy, elongated shoots when cultured on shoot bud regeneration medium (SRI) composed of 2 μM BAP, 2 μM KIN, 2 μM TDZ, and 0.5 μM NAA followed by multiple shoot regeneration (SRII) medium containing 2 μM BAP, 2 μM KIN, and multiple shoot elongation (SE) medium (0.5 μM of BAP + 0.5 μM of KIN). The presence of TDZ in combination with BAP and NAA in the SRI medium for one sub-culture cycle (10–14 days) significantly increases formation of multiple shoot buds per explant. Independent, healthy shoots obtained were selected for both in vitro rooting and grafting. Establishment of plantlets in the soil was highest (80–100%) in the case of in vitro rooted compared to grafted shoots (40%). The protocol appears to be competent to Agrobacterium-meditated transformation with ‘gus’ as a reporter gene. PCR analysis of the T0 and T1 progenies showed the presence and transmission of the transgene. We document here the regeneration and transformation of blackgram using cotyledons with wounded embryonic axes and the protocol appears to be suitable for genetic transformation of blackgram.

Role of TDZ in the Quick Regeneration of Multiple Shoots from Nodal Explant of Vitex trifolia L.—an Important Medicinal Plant

The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0μM). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with α-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0μM BA and 0.5μM NAA. The highest shoot regeneration frequency (90%) and maximum number (22.3 ± 0.2) of shoots per explant with shoot length of (5.2 ± 0.2cm) was recorded on MS medium fortified with 5.0μM TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5μM NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.

Establishment of a system for regeneration and transfor-mation in peanut (Arachis hypogaea L.) using somatic embryo

Currently, plant cell biotechnology has providing advanced method, especially systems for development of transgenic plants via Agrobacterium tumefaciens, to develop plant varieties with improved resistance to diseases and pests and better tolerance to abiotic stresses. In the case of peanut (Arachis hypogaea L.) methods for in vitro plant regeneration and gene transfer using callus tissue have been reported. In this study, peanut cultivar L12 was used for development of an effective system generation for somatic embryos, multiple shoots, rooting of regenerated plantlets and then for testing the efficiency of transformation using gus gene and somatic embyos. Our results showed that the MS medium supplemented with 20 mg/l of 2,4 D proved to be most suitable for somatic embryo induction (14.9 embryos/1 embryos); and the MS medium supplemented with 2 mg/l of BAP was most effective in multiple shoot regeneration; rooting of regerated shoots was succesfull in MS medium containing 0.3 mg/l of IBA; The use of the developed generation system for transfer of gus gene via Agrobacterium - mediated method has resulted gus positive transformed plants. Thus, the system could be asseful for the transfer of valuable genes in peanut.

English

An in vitro propagation protocol for an important ethnomedicinal plant, Tylophora indica(Burn.f) Merill has been developed. The effect of 0.5 to 3.0 mg/L benzo[a]pyrene (BAP) alone on in vitro multiple shoot production from leaf explants was studied. The maximum number of indirect regeneration of multiple shoots (7 shoots) was achieved from leaf explants on MS medium enriched with 2.5 mg/L BAP alone. Furthermore, large-scale shoot formation (25 shoots) was achieved by reported sub-culturing of leaf callus on shoot regeneration medium (MS + 2.5 mg/L BAP). The best root induction (97%) was achieved on MS medium enriched with indole-3-butyric acid (IBA) (0.5 mg/L). The plantlets were hardened off and more than 95% of survival plants were released to the shade house. The presence of alkaloids in vivo and in vitro samples was clearly detected.      Key words: Tylophora indica, leaf derived callus, regeneration, morphogenetic.

RETRACTED ARTICLE: Influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types

A comparative performance of two explants types (CN and Nodal) for their efficiency to induce multiple shoot regeneration in Clitoria ternatea has been carried out. Thidiazuron (TDZ) in different concentrations (0.05-2.5μM) was used as a supplement to the Murashige and Skoog's (MS) basal media. Explant type apart, two factors viz. concentration and exposure duration to TDZ played an important role in affecting multiple shoot regeneration. Cotyledonary node explants produced the best results at 0.1μM TDZ, while in nodal explants the highest rate of shoot formation was achieved on MS medium supplemented with 1.0μM TDZ. In both the explants, shoot multiplication increased when the regenerated shoots were subcultured on hormone free MS medium after 4weeks of exposure to TDZ. Among the two, cotyledonary node explants produced considerably higher number of shoots at a comparatively lower concentration of TDZ than nodal explants. The regenerated shoots rooted best on MS medium containing 1.0μM indole-3-butyric acid (IBA) and were successfully established in pots containing garden soil with 88% survival rate. All the regenerated plants showed normal morphology and growth characteristics.

In vitro multiple shoot regeneration from corm bud explant in ghet kachu, typhonium trilobatum schott - a medicinal aroid

An efficient in vitro regeneration protocol was developed in medicinal aroid, Ghetkachu (Typhonium trilobatum Schott) using field grown corm bud explant. Highest percentage (75 %) of direct multiple shoot regeneration obtained in MS media supplemented with 5.0 mgL-1BAP + 1.5mg L-1NAA. Callus formation occur (80 %) in MS media containing 0.5mgL-1BAP + 2.0mgL-1NAA. The appearance of calli was white, creamy white light green in colour and the texture of calli were soft, friable and semi hard and compact. Shoot regeneration (85 %) obtained from calli in MS medium having 5.0mgL-1BAP +1.0mgL-1NAA. The regenerated plantlets were successfully acclimatized with loamy fertile soil and survived cent percentage in natural condition. DOI: http://dx.doi.org/10.3329/bjsir.v47i2.11454 Bangladesh J. Sci. Ind. Res. 47(2), 211-216, 2012

An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers

Citrus jambhiri (rough lemon) is considered a major rootstock source for a number of Citrus species. A simple method for micropropagation from nodal segments is reported. Nodal segments of C. jambhiri were inoculated on MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin, and N6-(2-isopentenyl) adenosine (2iP). Maximum multiple shoot regeneration response (75 %) was observed with BAP at 3 mg l−1. Shoots were multiplied for 30 d on fresh medium with similar composition. A total of 67 % of the cultures showed multiplication with the optimum number of shoots (4.02) and height of shoots (1.81 cm) with BAP (3 mg l−1) alone. Maximum rooting response (87 %) was observed with naphthaleneacetic acid at 0.5 mg l−1. Transverse sections of shoot stems obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal segments), showed similar anatomies. Randomly amplified polymorphic DNA analysis confirmed that all the regenerated plants were genetically identical to their donor plant, suggesting absence of detectable genetic variation in the regenerated plantlets.

식물생장조절제 및 광원처리에 따른 헛개나무 줄기와 경정유래 신초의 기내증식

This study was conducted to examine effects of plant growth regulators and light resources on the formation of multiple shoot and plant regeneration of Hovenia dulcis var. koreana Nakai. Stem and shoot tip were cultured on MS medium or WPM supplemented with various plant growth regulators. At the single treatment, the highest shoot formation was obtained when stem explants were cultured on WPM supplemented with kinetin 1.0㎎ · L^-1. MS medium containing NAA 0.1 and TDZ 0.1 ㎎·L^-1 gave the best results for shoot induction rate and shoot growth in combination treatments. Of the BAP and kinetin tested, BAP 0.5 ㎎·L^-1 on WPM was found to be more effective for shoot growth from shoot tip. Under white fluorescent light treatment, shoot growth was much higher than blue, red LED treatments. Root induction from in vitro growth of plantlet was the best on WPM supplemented with 1.0㎎ · L^-1 IBA. The results suggest that selection of plant growth regulators and light resources could be important factor to achieve an efficient in vitro growth.

Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb

Stevia rebaudiana is a valuable medicinal plant species and it is being used for the treatment of diabetes. Currently, there is a high demand for raw material of this medicinal herb due to ever increasing diabetes disorder among the population. In order to meet the increased demand an efficient in vitro propagation of S. rebaudiana was established. Nodal explants collected from the field were cultured on MS basal medium fortified with different concentrations of BAP (0.5–3.0mg/l) and KIN (0.5–3.0mg/l) individually for shoot bud induction. In vitro derived nodal buds were cultured on MS medium supplemented with different concentrations (0.5–3.0mg/l) of BAP and KIN for multiple shoot bud regeneration. In the second experiment, in vitro derived buds were placed on MS medium supplemented with different concentrations of BAP (0.5–3.0mg/l) in combination with 0.5mg/l IAA or IBA or NAA for shoot bud multiplication. The highest frequency (94.50%) of multiple shoot regeneration with maximum number of shoots (15.69 shoots/explant) was noticed on MS medium supplemented with 1.0mg/l BAP. For large scale plant production, in vitro derived nodal bud explants were cultured on MS medium fortified with 1.0mg/l BAP, in which about 123 shoots/explant were obtained after three subcultures on the same media composition. Elongated shoots (>2cm) dissected out from the in vitro proliferated shoot clumps were cultured on half-strength MS medium containing different concentrations of NAA (0.1–0.5mg/l) and/or MS medium fortified with various concentrations (0.5–2.0mg/l) of auxins (NAA, IAA and IBA) for root induction. Highest frequency of rooting (96%) was noticed on half-strength MS medium augmented with 0.4mg/l NAA. The rooted plantlets were successfully transferred into plastic cups containing sand and soil in the ratio of 1:2 and subsequently established in the greenhouse. The present in vitro propagation protocol would facilitate an alternative method for rapid and large-scale production of this important antidiabetic medicinal plant.

In vitro Plant Regeneration in Brassica spp.

MS with different concentrations and combinations of hormones were used for in vitro multiple shoot regeneration in two varieties of Brassica juncea, namely, BARI Sarisha-11 and BARI Sarisha-16 and one variety of Brassica campestris, Tori-7. The highest percentage of responsive explants towards the regeneration of shoots was obtained on MS with 2.0 mg/l BAP, 0.2 mg/l NAA and 0.5 mg/l Kn in case of BARI Sarisha-11 and BARI Sarisha-16. For Tori-7, 3.0 mg/l BAP and 0.2 mg/l NAA was best for obtaining maximum number of shoots per explant. Among three varieties, BARI Sarisha-11 showed best response in terms of shoot regeneration as well as number of shoot per explant. Days required for induction of shoots was also recorded to be lowest in BARI Sarisha-11. Best root induction in BARI Sarisha-11 and BARI Sarisha-16 was achieved on hormone free MS. After proper hardening the in vitro regenerated plantlets were successfully transplanted into soil. Interestingly some of the in vitro regenerated shoots produced in vitro flowers on regeneration media as well as hormone free MS. Key words: Plant regeneration, Brassica juncea, Brassica campestris D. O. I. 10.3329/ptcb.v21i2.10235 Plant Tissue Cult. & Biotech. 21(2): 127-134, 2011 (December)

Role of growth regulators on in vitro regeneration and histological analysis in Indian ginseng (Withania somnifera L.) Dunal

An efficient, rapid and improved in vitro plant regeneration protocol has been established for Withania somnifera L. using shoot tip and nodal explants, excised from 15days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.5μM was most effective in proliferating apical and axillary buds. The highest regeneration frequency (95%) and number of shoots (36.1 ± 0.33) were obtained on MS medium fortified with BA (2.5μM) and NAA (0.5μM) from nodal segments. High frequency of rooting (100%) was obtained in in vitro raised shoots when transferred to half-strength MS medium supplemented with NAA (0.5μM). Histological sections revealed that additional shoot bud primordia were differentiated within the explants just underneath the suberized cells which appeared to be arrested in their development. The presence of additional bud primordia within the explants is thereby helpful to maximize the potential of this system. The regenerated plantlets with well developed shoots and roots were hardened successfully, established in earthen pots containing garden soil and maintained in greenhouse with 95% survival rate.

Effect of plant growth regulators on morphogenesis and forskolin production in Plectranthus barbatus Andrews

Plectranthus barbatus (syn. Coleus forskohlii) is the only known source of forskolin, a compound with a wide range of pharmacological activities. Here, an efficient protocol for adventitious root regeneration from leaf explants of P. barbatus was developed. Different concentrations of plant growth regulators individually and in combination were used to induce roots in vitro. Morphogenic responses and forskolin production varied depending on the concentrations of plant growth regulators added to the medium. Lower concentrations of auxins trigger callus proliferation while higher concentrations induced adventitious root regeneration. Of all the auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2 (2,4,5-trichlorophenoxy) propionic acid (2,4,5-TP), and 4-amino-3,5,6-trichloropicolinic acid (picloram) induced callus, whereas α-naphthaleneacetic acid (NAA), indole-3-acetic acid, and indole-3-butyric acid induced rhizogenesis. Use of picloram at 1.0 and 0.5 mg l−1 resulted in the formation of friable callus, and when combined with 0.5 mg l−1 6-benzylamino purine (BA), rhizogenic callus was produced. The cytokinins BA and kinetin produced a mixed response of multiple shoot regeneration, callus proliferation, and rhizogenesis. The maximum forskolin content of 1,178 mg kg−1 dry weight was found in root cultures initiated on Gamborg’s B5 medium supplemented with 0.5 mg l−1 NAA. The biosynthesis of forskolin was differentiation dependent, and rhizogenic cultures exhibited the maximum biosynthetic potential for forskolin.

High frequency in vitro shoot regeneration of Momordica balsamina, an important medicinal and nutritional plant

A protocol was developed for in vitro propagation by multiple shoot induction of M omordica balsamina ( Cucurbitaceae ), a climber with high medicinal and nutritional values. High frequencies of multiple shoot regeneration were achieved from auxillary bud of nodal explants. The bud explants were cultured on MS media supplemented with 1.0 mg/L benzyl amino purine (BAP) which stimulated proliferation of the bud meristems to form bud clusters having 6 to 8 co-efficient. The elongated shoots were sub-cultured for rooting on ½ MS media supplemented with 0.3 mg/L NAA and 0.2% activated charcoal. The plantlets raised in vitro were acclimatized in green house and successfully transplanted to natural condition with 70% survival. Direct organogenesis of explants and regeneration of M. balsamina offer a good opportunity to use tissue culture as a complementary tool for breeding and genetics for other applications. Key words : Momordica balsamina, regeneration, in vitro propagation, cucurbitaceae.

Influence of polyamines on shoot regeneration of sugarcane (<i>Saccharum officinalis</i>. L)

We studied the effect of polyamines (spermidine, putrescine and spermine) along with benzyladenine, kinetin and napthaleneacetic acid on multiple shoot regeneration of sugarcane. Murashige & Skoog medium containing a combination of benzyladenine (8.3 μM), kinetin (4.6 μM), napthaleneacetic acid (2.6 μM) and spermidine (68 μM) induced the maximum number of shoots (42 shoots/explant) compared to benzyladenine (8.3 μM), kinetin (4.6 μM) or napthaleneacetic acid (2.6 μM) alone or with putrecine (68 μM), spermine (32 μM) or combinations of polyamines. Plantlets raised were successfully transplanted in soil with a 90% survival rate.