Optimization of Plant Growth Regulators and Bioreactor Systems for Efficient In Vitro Shoot Multiplication and Elongation of Amorphophallus muelleri Blume

Shafira taro (Colocasia esculenta var. antiquorum) is a type of small-tubered taro, also known as Japanese taro, which is traded internationally. This study aims to examine the effect of various concentrations of Thidiazuron (TDZ) and Benzyl Amino Purine (BAP), as well as the combination of Thidiazuron with Naphtalene Acetic Acid (NAA) and BAP with NAA, on the multiplication of Shafira taro shoots through in vitro culture techniques. The method used in this study uses in vitro culture techniques, which are modern plant propagation science known in the world of biotechnology and allow controlled propagation and improvement of plant species. An essential component of this technique is using plant growth regulators (PGRs), critical for modulating various physiological processes in plant cells and tissues. PGRs promote growth and differentiation, optimize secondary metabolite production, and increase plant resistance. This study used a combination of main PGR treatments (TDZ, BAP, and NAA) with a total of 13 (thirteen) treatment combinations using a randomized block design, namely: k0: Control, t1: TDZ 1 ppm, t2: TDZ 2 ppm, t3: TDZ 3 ppm, b1: BAP 1 ppm, b2: BAP 2 ppm, b3: BAP 3 ppm, t1n1: TDZ 1 ppm + NAA 0.5 ppm, t2n1: TDZ 2 ppm + NAA 0.5 ppm, t3n1: TDZ 3 ppm + NAA 0.5 ppm, b1n1: BAP 1 ppm + NAA 0.5 ppm, b2n1: BAP 2 ppm + NAA 0.5 ppm, b3n1: BAP 3 ppm + NAA 0.5 ppm. Each treatment was repeated thrice, with three tissue culture bottles per repetition, resulting in 117 tubes containing one explant. The results showed that the highest number of roots, leaves, and shoots was achieved with 1 ppm BAP, while the optimal fresh weight and shoot height were obtained with 2 ppm BAP. Treatments involving combinations of TDZ, TDZ + NAA, and BAP + NAA did not produce significant results for shoot multiplication.

Platycodon grandiflorum (Bell flower) is an important plant that has traditionally been used as herbal medicine for the treatment of cough, phlegm, sore throats, lung abscesses, chest pains, dysuria, and dysentery. The present study was initiated to investigate the feasibility of inducing shoot and root organogenesis in cultured explants of P. grandiflorum in a range of culture media and through use of various plant growth regulators (PGRs). The plantlets (Stem containing one node) were isolated and cultured on different concentrations of Murashige and Skoog (MS) medium supplemented with PGRs. We found that proliferation and elongation of shoots and roots could be achieved on ¼ MS for P. grandiflorum with wild and green petals and on ⅛ MS for P. grandiflorum with double petals. The highest levels of development and elongation of adventitious shoots and roots were observed when petal explants were cultured on ¼ MS (pH 3.8) supplemented with 5% sucrose. Increasing the agar concentration reduced shoot growth and rooting potential; nevertheless, the highest number of shoots and roots was observed on 0.6% agar. In the case of growth regulators, ¼ MS supplemented with 1 mg L-1 6-benzylaminopurine (BA) was found to be best for shooting, although higher concentrations of BA tended to reduce shoot and root elongation. The highest number of shoots was achieved on 0.5 mg ․ L-1 thidiazuron (TDZ) from double petal explants grown on ⅛ MS. However, root and shoot elongation were found to decrease when TDZ concentrations were increased. Low concentrations of kinetin, naphthalene acetic acid, indole acetic acid, and 3-indole butyric acid induced shoot and root proliferation and elongation. Taken together, our study showed that low concentrations of PGRs induced the greatest root formation and elongation, showing that the optimal concentration of PGRs for shoot proliferation was species-dependent.

ADVENTITIOUS SHOOT FORMATION ON TEAK (TECTONA GRANDIS L.F.) CALLUS CULTURES DERIVED FROM INTERNODAL SEGMENTS

SummaryAn efficient in vitro regeneration procedure using thidiazuron (TDZ) has been developed to allow high frequency, multiple shoot induction from cotyledonary node explants of cluster bean (Cyamopsis tetragonoloba). Shoot bud induction occurred on Murashige and Skoog (MS) medium after 4 weeks in the presence of TDZ, followed by transfer onto shoot multiplication and elongation media containing MS salts, B5 vitamins, and different combinations of auxins and cytokinins. Multiple shoots were induced at all levels of TDZ in the medium, but the best proliferation capacity occurred at 5 µM TDZ. Combinations of auxins and cytokinins showed a stimulatory effect on shoot multiplication and also on the length of the newly formed shoots. Maximum shoot induction [i.e., the highest number of shoots (16.0 ± 0.94) per explant] was obtained on agar-solidified medium containing 5 µM benzyladenine (BA) with 0.5 µM indole-3-acetic acid (IAA). Rooting of in vitro-regenerated shoots was achieved in ex vitro conditions by a pulse treatment with 300 µM indole-3-butyric acid (IBA) for 15 min. Rooted plantlets were transferred to soil where 70 – 75% attained sexual maturity and produced viable seeds under greenhouse conditions. The present regeneration system is efficient and can be used in various in vitro manipulation studies.

The pear (Pyrus spp.) is one of the most important temperate fruit crops. A complete protocol for adventitious shoot regeneration was developed from the leaves of four pear varieties grown in vitro: Abbe Fetel, Yali, Packham’s Triumph and Aikansui, and the Chinese rootstock variety Duli. Shoot explants were collected from the field and cultured in vitro in Murashige and Skoog (MS) media supplemented with 1.0 mg·L−1 6-benzylaminopurine (BA) and 0.1 mg·L−1 indole-3-butyric acid (IBA). After four weeks, leaf explants of all 5 varieties grown in vitro were excised and cultured in MS media supplemented with 0.0 mg·L−1, 0.2mg·L−1, 0.5 mg·L−1, 1.0 mg·L−1 and 2.0 mg·L−1 naphthaleneacetic acid (NAA) and 5.0 mg·L−1 BA or with 1.0 mg·L−1, 2.0 mg·L−1 and 4.0 mg·L−1 thidiazuron (TDZ). The cultures were maintained in darkness for 21 days for shoot induction in the shoot induction medium (IM), then transferred to the shoot expression medium (EM) in 1.0 mg·L−1 TDZ without any auxins and kept in a growth room at (25±2)°C under a 16/8 h light/dark photoperiod regime for 8 weeks. Finally, the shoots were transferred to the MS shoot elongation medium (SEM) supplemented with 0.2 mg·L−1 BA, 0.1 mg·L−1 IBA and 0.2 mg·L−1 gibberellic acid (GA3). A combination of TDZ and NAA had a significant effect on the number of shoot regenerations in all 5 tested varieties. The maximum mean number of shoots and maximum number of shoots per leaf obtained from Yali variety were 11.8 (P⩽0.001) and 22, followed by Aikansui with 6.6 (P⩽0.001) and 4.6, and Duli with 8 (P⩽0.001) and 12, all arising from the combination of 0.2 mg·L−1 NAA with 1.0 mg·L−1 TDZ. For Packham’s Triumph and Abbe Fetel, the maximum mean number of shoots and maximum number of shoots per leaf were 5.6 (P⩽0.001), 4.8 and 8 (P⩽0.001), and 11, respectively, from the combination of NAA (1.0 mg·L−1) and TDZ (2.0 mg·L−1). Abbe Fetel was the only variety which produced significantly higher adventitious shoots from the two different combinations of 1.0 mg·L−1 NAA and 5.0 mg·L−1 BA (P⩽0.05), and 2.0 mg·L−1 NAA and 5.0 mg·L−1 BA (P⩽0.01). Some of the most prominent problems associated with shoot proliferation and regeneration were also observed and discussed in this paper.

Adventitious shoot induction and elongation was compared between root and petiole explants of Kentucky coffeetree (Gymnocladus dioicus L.) explants treated with a factorial combination of benzylaminopurine (BA) and thidiazuron (TDZ). Petiole explants initiated more adventitious shoots compared to root explants. Up to 83% of petiole explants initiated shoots compared to 67% of root explants. Maximal shoot induction was approximately 12 or five shoots per responding explant for petiole and root explants, respectively. For both explant types, TDZ was more effective than BA for shoot induction. There was an interaction between BA and TDZ on shoot induction in petiole explants, with the greatest percentage of explants forming shoots and the highest number of shoots initiated on the combination of 0.5 μM TDZ plus 10 μM BA and 1.0 μM TDZ plus 5 or 10 μM BA. In contrast, increasing concentrations of BA inhibited shoot initiation in root explants with and without TDZ. While BA inhibited shoot initiation i...

The regeneration potential of excised aspen (Populus tremula L.) roots cultivated in liquid medium, as affected by plant growth regulators and by the position of the isolated root explant on the main root, was investigated. The effect of various levels of benzyladenine (BA) and thidiazuron (TDZ) on bud regeneration in root explants was studied. TDZ in the medium had a marked effect on bud development as compared with BA, inducing a tenfold increase in the number of buds regenerated from various root explants. TDZ enhanced both root and root-borne shoot biomass production but reduced further shoot development and elongation. The position of the isolated root sections on the main root affected regeneration, the proximal sections further away from the root tip producing the highest number of buds per explant in both BA and TDZ treatments. Buds regenerated in close proximity to the site of lateral roots in BA-treated roots, while in TDZ-treated root sections, the buds formed all over the root regardless of the presence of lateral roots. The buds developed from inner cortical and sub-epidermal cell layers, disrupting the epidermis and the inner layers. Root biomass production and growth was greatly enhanced in well-aerated bioreactor culture in the presence of 4.5×10-2 μM TDZ. A high number of the root-borne shoots could be rooted and converted to plantlets. However, while shoots regenerated in a medium with BA rooted well in a growth regulator-free medium, shoots formed in a medium with TDZ required auxin for rooting. Roots cultured in the presence of ancymidol, a gibberellin biosynthesis inhibitor, regenerated non-hyperhydric bud clusters and hyperhydric shoots. These were separated mechanically, subcultured to growth and rooting medium and transplanted ex vitro resulting in phenotypically true-to-type plantlets. The potential of liquid cultures for aspen shoot biomass production from roots is discussed.

Costus speciosus is being rapidly eliminated in its natural habitats in India and driven to a nearly threatened degree of extinction because of non-selective collection and over-exploitation. The commercial cultivation of C. speciosus is impeded owing to its poor seed viability, reduced germination rate, and weak rooting potential of vegetative cuttings. As costus could not be multiplied rapidly by conventional propagation methods, in-vitro regeneration technology was experimented with to standardize explants in addition to the combinations of plant growth regulators (PGRs) to fortify the growing media that helps to produce the enormous number of plants in a shorter duration. Several kinds of explants viz., leaves and nodes were cultured into the Murashige and Skoog (MS) medium reinforced with different combinations and doses of PGRs. A higher frequency (51.67 ± 1.7%) of the regenerated adventitious shoot was obtained from nodal segments cultured onto MS medium fortified with 0.5 mg/l Thidiazuron (TDZ) and 2.0 mg/l of 6-benzyl amino purine (BAP). TDZ in combination with BAP and Indole-3-acetic acid (IAA) exhibited a remarkable effect on shoot multiplication and elongation. Further, MS medium with 0.5 mg/l TDZ, 1.0 mg/l 2, 4-dichlorophenoxyacetic acid, and 0.5 mg/l IAA yielded a great range of callus initiation (86.66 ± 1.6%) from leaf explants. Leaf-derived callus results in the greatest shoot regeneration (56.66 ± 1.7%) response on TDZ (0.5 mg/l) and BAP (1.0 mg/l) reinforced MS medium. Elongated shoots from node and callus cultures when inoculated with the MS medium reinforced along with 1.0 mg/l of IAA and 0.5 mg/l of Indole-3-butyric acid (IBA) produced optimum rooting (88.33 ± 1.66%). The rooted young plantlets were effectively acclimatized to the environmental climate and their greenhouse survival rate was 85%.

The wilt disease caused by Ralstonia solanacearum and the leaf spot disease caused by Phyllosticta sp. are significant constraints in ginger cultivation as they can lead to crop failure. One approach to eliminating these diseases is to use disease-free ginger plantlets obtained through tissue culture propagation. This study investigated the influence of plant growth regulators, i.e., Benzyl Adenine (BA) and Thidiazuron (TDZ), on the in vitro multiplication of large white ginger shoots. The tested treatments included combinations of BA (0, 1, 2, 3 mg L-1) and TDZ (0, 0.1, and 0.2 mg L-1), with ten replicates each. A complete randomised factorial experimental design was employed. The observed variables were shoot height, number of shoots, number of leaves, and number and length of roots at 2, 4, 6, and 8 weeks of age. The results indicated an interaction between TDZ and BA for shoot number and root length. The highest numbers of shoots were obtained after eight weeks using 0.1 mg L-1 TDZ alone without BA. Meanwhile, the longest roots were obtained after eight weeks using a specific combination of TDZ and BA concentrations. Based on this study, we proposed a strategy to implement this protocol to induce the formation of shoots, leaves, and roots in a multistep tissue culture propagation.

A procedure for multiple shoot formation from cotyledonary node explants of faba bean (Vicia faba L.cv.S.Ghdar) cultured on MS medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced a higher number of shoots than with either cytokinin alone. The highest number of shoots was obtained when explants from 7-day-old seedlings were cultured on MS medium supplemented with TDZ and BA (2 mgl−1 each) for 31 days before transfer to hormone-free MS medium for elongation. Shoots produced in vitro were rooted on half-strength agar-solidified MS basal medium or with 0.25 or 0.5 mgl−1 naphthalenacetic acid (NAA) prior to transfer to green house conditions. This procedure was found to be applicable to seven other cultivars of faba bean from widely diverse provenances. Thus, it can be advantageously applied to the production of transgenic faba bean plants.

A procedure for multiple shoot formation from cotyledonary node explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. Explants on medium with TDZ in combination with BA produced higher numbers of shoots than with either cytokinin alone. The highest number of shoots (7.8 to 9.8 shoots per explant) was obtained when explants from 4 to 10 day-old seedlings were treated with a combination of 10 or 15 µM BA and 0.5 or 1.0 µM TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 5.8 to 7.2 shoots per explant by cutting through the cotyledonary node prior to culture. Histological studies indicated that the shoots were formed from actively dividing cells located at the axillary bud region. Shoots formed roots in half strength woody plant medium (WPM) supplemented with 10 to 200 µM indole-3-butyric acid (IBA) cultured for 15 days prior to transfer to greenhouse medium.

A procedure for multiple shoot formation from somatic embryo explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. TDZ in combination with BA produced more shoots than either treatment alone. The highest number of shoots (3.3 to 3.4 shoots per explant) was obtained from partially desiccated and wounded explants treated with a combination of 5 or 10 μM BA and 0.5 or 1.0 μM TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 1.5 to 3.2 shoots per explant by cutting through the cotyledonary node prior to culture. In addition, the frequency of explants forming shoots was increased by desiccation of somatic embryo explants to approximately 50% moisture and by using somatic embryos with two well formed cotyledons as explants.

SummaryAn efficient in vitro protocol using dormant buds as explants was standardised for three Morus (mulberry) species: M. alba, M. indica, and M. laevigata. Explants were collected from field-grown plants and cultured on 1.0_ Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of phytohormones. Thidiazuron (TDZ) at 0.1 mg l-1 or benzylaminopurine (BAP) at 1.0 mg l-1, added separately, gave the best rates of shoot initiation and shoot induction, and required less time for bud sprouting in all three species. Basal MS medium resulted in the lowest rate of shoot induction and the longest duration for shoot initiation. Shoot multiplication was induced by a combination of 0.5 mg l-1 BAP, 0.5 mg l-1 kinetin (Kn), and 0.1 mg l-1 indole-3-acetic acid (IAA). Shoot proliferation and the elongation of adventitious shoots were observed using 1.0 mg l-1 BAP plus 0.2 mg l-1 gibberellic acid (GA3). The rooting percentage was highest (90%) on 0.5_ MS medium supplemented with 0.5 mg l-1 indole-3-butyric acid (IBA), which was more effective than rooting (70%) on 1.0 mg l-1 _-naphthaleneacetic acid (NAA). Well-rooted plantlets exhibited normal growth under greenhouse conditions, with a 98% survival rate. Plants derived from this multiple shoot induction protocol showed no apparent differences in phenotype in the greenhouse. This protocol would be of use for large-scale propagation of mulberry to support ex situ conservation.

SummaryThe effect of thidiazuron (TDZ) on the shoot production capacity of preconditioned explants of Castanea sativa Mill. was evaluated. Embryonic axes of chestnut were aseptically germinated for 14.d in Murashige and Skoog (1962) basal medium supplemented with 0.1.mg l±1 TDZ or 1.mg l±1 benzyladenine (BA) (preconditioning medium), and hypocotyl, epicotyl and cotyledonary node explants were then cultured on shoot-induction medium (basal medium containing 0.01.mg l±1 naphthaleneacetic acid in combination with different TDZ concentrations). After four weeks of culture the explants were transferred for a further eight weeks to media with low BA concentrations and no TDZ. Shoots were produced only by cotyledonary node explants which contain preformed meristematic tissue. Best shoot production, in terms of the proportion of explants producing shoots and shoot productivity per explant, was achieved by BA-preconditioned explants exposed to 1±2.mg l±1 TDZ. Fewer TDZ-preconditioned explants produced shoots, and more produced only callus. An histological study showed preconditioned explants to have an altered meristematic axillary region, with meristematic cell proliferation giving rise to expanded, flattened apical meristems and reduced leaf primordium differentiation. Upon shoot-induction treatment the proliferating meristems orginated multiple shoot buds without an intervening callus phase. Shoot elongation was achieved by culturing the original explants in Gresshoff and Doy (1972) medium supplemented with 0.05.mg l±1 BA. Shoots longer than 10.mm were subcultured separately to initiate clonal shoot cultures that were proliferated following the outgrowth of axillary shoots. Rooting frequencies greater than 75% were achieved by 16 of the 21 clones evaluated. Rooting capacity was not influenced by either the preconditioning medium or the shoot induction medium, but considerable variation was observed between clones (genotypes).

The clonal propagation with nodal cultures of the medicinal endemic plant species Astragalus trojanus Stev. was aimed in this study. Seeds were germinated in Murashige and Skoog (MS) medium, and the nodal segments obtained from seedlings were used as an explant. Firstly, the effects of MS and (Woody Plant Medium) WPM nutrient media on shoot multiplication were investigated. The explants were cultured in MS or WPM nutrient media containing at different concentrations (0.2, 0.5, 1.0, 2.0 mg L -1 ) 6-benzyl amino purine (BAP). After 4 weeks from culture, the highest number of shoots (2.80 number/explant) was obtained in WPM medium containing 0.5 mg L -1 BAP. Nodal segments were cultured in WPM medium containing different concentrations of BAP, thidiazuron (TDZ), kinetin (KIN) (0.2, 0.5, 1.0, 2.0 mg L -1 ) to determine the effect of cytokinin source and concentrations on shoot multiplication. At the end of 4 weeks, it was determined that TDZ and KIN had similar effect on number of shoots. The highest number of shoots (2.09 number/explant) was obtained from WPM media containing BAP. Regenerated shoots were transferred to WPM media containing 1 or 3 mg L -1 indole-3-butyric acid (IBA), naphtalene acetic acid (NAA) or indole-3-acetic acid (IAA) for rooting. After 4 weeks from culture, the most root number (4.33 number/explant) were obtained from the WPM medium containing 3 mg L -1 IAA, while the highest rooting percentage was obtained from WPM medium containing 3 mg L -1 IBA with 80%. Rooted seedlings were transferred to plastic cups containing peat and perlite in a ratio of 1:1 and successfully acclimatized. Plant culture was completed at 15 weeks. The effective clonal propagation method was described for the endemic species Astragalus trojanus Stev.