Multiple Shoot Formation Research Articles

UTILIZAÇÃO DA BACTÉRIA Rhodococcus fascians NA OTIMIZAÇÃO DO PROCESSO DE FORMAÇÃO DE BROTOS DE ACÁCIA NEGRA

A acácia negra (Acacia mearnsii) é cultivada por pequenos produtores na região Sul do Brasil. Esta espécie produz madeira, celulose e taninos. Devido a essa importância, é preciso estabelecer técnicas de propagação desta espécie em grande escala. Uma técnica que pode possibilitar a formação de brotos múltiplos consiste em utilizar a bactéria Rhodococcus fascians. Quando plântulas de acácia negra germinadas in vitro são infectadas, no lugar do ápice se desenvolvem galhas formadas por centros meristemáticos múltiplos e primórdios de gemas. Para favorecer o alongamento dessas gemas, galhas foram separadas das plântulas e cultivadas em meio de MURASHIGE e SKOOG (1962) contendo ¾ da concentração dos sais, 20 g.L-1 de sacarose e 7 g.L-1 de ágar. Três experimentos foram realizados. No primeiro foi testada a adição ou não de carvão ativado (CA) (2 g.L-1); no segundo foram testadas a ausência e três concentrações (0,29; 1,44 e 2,89 µM) de ácido giberélico (GA3) e no terceiro a ausência e três concentrações (1,0; 2,0 e 5,0 µM) de ácido 2,3,5-triiodobenzóico (TIBA). A adição de CA no meio de cultura não aumentou a formação de brotos, porém inibiu a indução de raizes. Já no segundo experimento, a suplementação de 1,44 µM de GA3 ao meio de cultura provocou a formação de um maior, porém não significativo, número médio de brotos por galha. No terceiro experimento, foi possível observar que as concentrações de 2 e 5 µM de TIBA possibilitaram uma formação de brotos significativamente maior que o meio de cultura desprovido de TIBA.

Micropropagation of Endangered Species Daphne cneorum

A new protocol for micropropagation of endangered Daphne cneorum through multiple shoot formation has been developed. Two different types of explants (dormant apical buds and in vitro seed-derived young seedlings) from plants in two different localities were used for the initiation of multiple shoots on agar woody plant medium (WPM) with 0.2 mg dm−3 benzylaminopurine (BAP), 0.1 mg dm−3β-indolebutyric acid (IBA), 200 mg dm−3 glutamine, and 200 mg dm−3 casein hydrolysate. From 10 seeds only one germinated and the multi-apex culture bearing 12 shoots sprouted out from in vitro seed-derived young seedling. After 6-month cultivation 35 multi-apex cultures were achieved from in vitro seed-derived young seedling. On 1/3 strength WPM medium supplemented with 2.83 mg dm−3 IBA 50 % of cultures (clusters of 3 – 5 shoots) rooted but no rooting occurred in the presence of α-naphthaleneacetic acid (NAA). The rooted plantlets were acclimatized for 4 weeks in the greenhouse and then transferred into natural conditions. The plants successfully survived the winter and flowered.

Micropropagation of an Endangered Orchid Anoectochilus formosanus

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

산마늘 다신초 증식과 인경형성에 효율적인 생물반응기 배양방식

본 실험은 산마늘의 산초 증식과 인경 형성에 적합한 생물반응기 배양방식과 인경 형성시 배양방식에 따른 당 대사를 구명하고자 실시되었다. 다신초 증식에는 생물반응기에 망을 걸고 배지에 절편체를 접하게 배양한 RC와 MRC (13-15개)에서 가장 좋은 결과를 얻었다. 인경형성과 비대에는 간헐적으로 배지를 공급해준 E&FS에서 93.4％의 인경형성이 이루어졌고 크기에 있어서도 균일하였다. RC와 MRC에서 형성된 인경은 뿌리가 무성하였으며 인경의 크기도 균일하지 않았다. 배양방식별로 수확한 인경내 유리당 함량은 전반적으로 E&FS.에서 낮았던데 반해 전분 함량은 높았다. 배지내 sucrose, glucose와 fructose는 인경 비대 시기에 감사되었는데 이는 첨가한 sucrose가 가수분해 되기도 전에 glucose나 fructose와 함께 식물체로 바로 이용됨을 보여주는 것이었다. A suitable bioreactor culture system for shoot proliferation and bulblet formation of Allium victorialis var. platyphyllum Makino was established. Uptake of soluble carbohydrates in different bioreactor culture systems was also analyzed during the entire culture period. Optimal conditions for multiple shoot formation were determined in raft culture (RC) and modified raft culture system (MRC) (13-15 per explant) in which the explants were placed on a net contacting liquid medium. For bulblet formation and enlargement, 93.4％ of shoot clumps formed bulblets at the basal part. Furthermore, they were uniform in size when cultured with ebb & flood system (E&FS). Bulblets harvested from RC and MRC showed vigorous rooting, however, their growth was not uniform. Whereas soluble carbohydrate contents in the bulblets cultured in E&FS were low, starch content was high. Sucrose, glucose and fructose concentrations in the medium of E&FS culture system decreased as bulblet formation and enlargement proceeded, suggesting that external sucrose is taken up to by the cells before it is hydrolyzed.

Alocasia cadieri Chantrier의 기내번식

본 실험은 Alocasia cadieri Chantrier를 기내배양하여 일시에 균일한 식물체를 대량생산하기 위하여 실시하였다. Alocasia의 경정을 TDZ 0.1mg/L가 첨가된 배지에 배양하였을 때 신초증식이 가장 높았으며, 형성된 신초에서 신초의 증식은 TDZ과 NAA가 각각 0.5mg/L첨가된 배지에서 효과적이었다. 형성된 신초의 발근은 IBA또는 NAA가 첨가된 배지보다는 활성탄 2.0g/L가 첨가된 배지에서 양호하였으며, 소식물체의 순화는 perlite : vermiculite가 1:1로 혼합된 용토 또는 vermiculite가 적합하였다. In order to micropropagate uniform plantlets of Alocasia cadieri Chantrier in vitro, the shoot tips were cultured on media containing various concentrations of BA and thidiazuron (TDZ). Multiple shoot formation from shoot tips was very effective on medium containing 0.1mg/L TDZ. The formed shoots from shoot tips were separated into a shoot, and cultured on media with BA, TDZ, and NM combination for proliferation. The shoots were multiplied very vigorously on medium with 0.5mg/L TDZ and 0.5mg/L NAA. The rooting and growth of multiplied shoots were more effective on medium with 2.0g/L activated charcoal, rather than those with IBA and NAA. Rooted plantlets show high survival in soil mixed with perlite 1: vermiculite 1 or vermiculite alone.

Micropropagation of Spilanthes acmella L., a bio-insecticide plant, through proliferation of multiple shoots.

Spilanthes acmella L. was successfully micropropagated using axillary buds as explants. The aseptic axillary buds formed multiple shoots within five weeks when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l N 6 -Bezyl adenine (BA). The addition of Indole-3-buty ric acid (IBA) had no significant effect on the multiple shoots formation of this plant. MS medium supplemented with 0.5 mg/l BA was sufficient for the proliferation of rooted multiple shoots of S. acmella L. First subculturing of the in vitro individual shoots in the same proliferation medium could double the formation of multiple shoots.

지리오가피 (Acanthopanax chiisanensis) 체세포배의 건조처리를 통한 식물체 증식

지리오가피의 체세포배로부터 식물체재생을 위한 효율적인 방법을 개발하였다. 체세포배의 형태형성에 미치는 식물생장 조절물질의 영향을 조사하고자, 접합자 배 유래의 배발생 세포를 MS배지에 현탁배양하고, 이로부터 얻어진 자엽시기의 배를 배양접시에서 건조처리 (0∼72 h)한 후, NAA, BA, GA<TEX>$_3$</TEX>(0∼0.5mg/L)가 첨가된 MS배지에 배양하였다. 그 결과, 체세포배를 72시간 동안 배양접시에서 건조처리 후에 0.5mg/L NAA, 0.5mg/L BA 또는 0.5mg/L NAA, 0.5mg/L BA 0.5mg/L GA<TEX>$_3$</TEX>가 첨가된 MS배지에 6주간 배양하였을 때, multiple shoot가 100%로서 가장 높게 유도됨을 관찰 할 수 있었다. 가장 높은 식물체의 재생율 (29.1%)은 체세포배를 72시간 건조처리 후에,0.1mg/L NAA, 0.1mg/L BA그리고 0.1mg/L GA<TEX>$_3$</TEX>가 첨가된 MS배지에 6주간 이식한 후, 이를 다시 1.0mg/L GA<TEX>$_3$</TEX>가 첨가된 1/3MS배지에 6주간 배양하였을 때 이루어졌다. 유식물체를 vermiculite와 sand가 1:1로 첨가된 plastic pot 이식하였을 때 생존율은 약 70%로서 나타났다 이상의 결과는 지리오가피의 계량생산에 활용될 수 있을 것으로 기대된다. An efficient method of plant regeneration from Acanthopanax chiisanensis somatic embryos was developed. Cotyledonary somatic embryos were obtained in liquid Murashige and Skoog (MS) medium from embryogenic cell suspension cultures. They were desiccated for 0 to 72 hr and then cultured on MS medium containing NAA, BA, GA<TEX>$_3$</TEX>, (0-0.5mg/L). The highest multiple shoots formation (100％) was obtained from 72 hr desiccated somatic embryos on ifs medium with 0.5mg/L NAA＋0.5mg/L BA or 0.5 mg/L NAA＋0.5mg/L BA＋0.5mg/L GA<TEX>$_3$</TEX> after 6 weeks culture. Plant conversion from multiple shoots was not high. The highest plant conversion from multiple shoots was obtained on 1/3MS medium with 1.0mg/L GA<TEX>$_3$</TEX>. Converted plantlets were transferred to ex vitro condition and the highest survival rate (70％) of the plantlets was obtained on plastic pots containing vermiculite and sand. These results indicate that micropropagation procedure can be applied for an efficient mass propagation of Acanthopanax chiisanensis.

In vitro clonal propagation of an adult tree of neem (Azadirachta indica A. Juss.) by forced axillary branching

In vitro clonal propagation of a 50-year-old neem tree through axillary shoot proliferation was achieved. Nodal explants collected between March and May gave the best shoot proliferation response (80% aseptic cultures). 1/2 MS (major inorganic salts reduced to half strength) medium was required initially for the establishment of nodal segment cultures. Multiple shoot formation occurred in the cultures of 1/2 MS+BAP (1μM) + GA3 (0.5μM). Number of shoots enhanced further on transfer of cultures to 1/2 MS+BAP (1μM) + CH (500mgl−1). However, both the media did not support shoot growth and the shoots remained compact and stunted. Therefore, for elongation and recurrent shoot multiplication, full MS+BAP (1μM) + CH (250mgl−1) medium was used where the shoots elongated well, and could be multiplied through single node segment cultures at a rate of 7–8-fold every 5 weeks on the fresh medium of the same composition and this rate of shoot multiplication was maintained for almost 5 years. On 1/4 MS+IBA (0.5μM), the shoots could be readily rooted with a frequency as high as 82%. Micropropagated plants were established in soil with >87% survival.

MICROPROPAGATION OF THE ARACEAE ORNAMENTAL PLANTS

Five species of Araceae plants were successfully cultured via in vitro methods. Multiple shoots were induced from four Araceae species, Alocasia sanderina, Colocasia antiquorum, Gonatanthus pumilus and Syngonium podophylum., when the axillary buds were cultured in Murashige and Skoog medium (MS) supplemented with 2.0 mg l -1 N 6 -benzyladenine (BA) and 0.5 mg l -1 indole-3-butyric acid (IBA). Basic MS medium supplemented with only 2.0 mg l -1 BA was found to be the most suitable medium for induction of multiple shoots in Caladium hortulanum and Caladium bicolor 'Florida Crown'. However, the same medium did not induce multiple shoot formation for Caladium humboldtii. The in vitro shoot microcuttings of these five Araceae species produced roots when cultured in basic MS medium to form complete plantlets. All the rooted in vitro plantlets (100%) survived when transferred to the outside environment after acclimatization for two weeks in an incubator at 25°C.

Multiple Shoot Regeneration from Immature Embryo Explants of Papaya

A simple and rapid method for multiple shoot formation in vitro from immature embryo axis explants of Carica papaya L. cvs. Honey Dew, Washington and Co2 is described. Multiple shoot regeneration was achieved by culture of the explants on modified Murashige and Skoog (MS) medium supplemented either with thidiazuron (TDZ; 0.45-22.7 &mu;M) or a combination of benzylaminopurine (BAP; 0.2 - 8.84 &mu;M) and naphthalene acetic acid (NAA; 0.5 - 2.64 &mu;M). Highest frequency of shoot regeneration occurred on medium supplemented either with 2.25 &mu;M TDZ or a combination of BAP (4.4 &mu;M) and NAA (0.5 &mu;M). Composition of the basal media influenced the frequency of multiple shoot initiation. Stunted shoots regenerated at 4.5 &mu;M and higher concentrations of TDZ. Such shoots could, however, be elongated by transfer to medium containing 5.7 &mu;M GA3. Rooting of the regenerated shoots was achieved in presence of indolebutyric acid (IBA; 4.92 - 19.68 &mu;M), however, least response was in presence of 14.7 &mu;M IBA. Rooted plants were hardened and transferred to pots.

In vitro regeneration of Ensete ventricosum from zygotic embryos of stored seeds

Plant regeneration through in vitro germination and the production of adventitious shoots from zygotic embryos was investigated using stored seeds of two enset genotypes (Mariya and Oniya), as germination of intact seeds could not be achieved. The seeds were decontaminated before and after water pretreatment and the embryos cultured on MS medium. Germination was studied with vertical and horizontal embryo orientations, on three media compositions: MS medium, MS with 0.5 mg l − 1 BA + 0.2 mg l − 1 lAAand MS with 0.5 mg l − 1 BA + 0.2 mg l − 1 2,4-D. Treating seeds of the Oniya clone with distilled water before decontamination and excision of embryos caused extensive contamination of cultured embryos. Decontamination without water pretreatment showed only about 5% contamination. Contamination was low with Mariya but no germination occurred. Horizontal embryo orientation resulted in better germination for Oniya. Supplementing MS medium with BA and IAA improved germination of the excised embryos (44%) and promoted formation of multiple shoots and simultaneous initiation of callus in some embryos. Subculturing the callus on the same medium composition (MS with BA + IAA) resulted in more and earlier initiation and proliferation of organogenic callus in the dark than other media compositions. Adventitious shoots were regenerated in the light on MS medium lacking plant growth regulators. Proliferation of a watery and friable callus was better when 2,4-D (0.2 mg l − 1 ) was added in the presence of BA and IAA during subculturing.

Multiple Benzyladenine Applications Increase Shoot Formation in Nandina

Abstract A study conducted from 2000 to 2002 determined the effects of multiple applications of benzyladenine (BA) applied at different concentrations on shoot formation, whole plant size, and phytotoxicity in Nandina domestica Thunb. In 2000, shoot counts increased in ‘Harbour Dwarf’ by up to 107% and 480% as the number of weekly BA applications at 2500 and 5000 ppm, respectively, increased to five. Likewise, the number of shoots per plant increased as BA rate increased. In response to five weekly foliar BA applications in 2001 and 2002, the number of new shoots on ‘Harbour Dwarf’ increased linearly 10-fold over the control as BA rate increased to 5000 ppm in 1250 ppm increments. In 2002, shoot formation increased linearly by up to six additional shoots per plant with increasing BA rate in ‘Harbour Dwarf’ and quadratically by up to two additional shoots per plant in ‘Firepower’ by 90 days after initial treatment (DAT) with five applications of up to 5000 ppm BA. Shoot formation in the tall, upright cultivars ‘Royal Princess’ and ‘Moyer's Red’ also increased linearly with increasing BA rate, by two and &amp;lt;1 shoot per plant, respectively, in 2002 only. Five foliar applications of 2500, 3750, and 5000 ppm BA injured upper, immature foliage, but did not adversely affect plant appearance by 90 DAT. Treatment effects on plant size were minimal.

Multiple shoot formation in Hypericum perforatum L.and hypericin production

Hypericum perforatum is a traditional medicinal plant with wound healing and antidepressive properties. Among the secondary compounds of interest is hypericin, a naphtodianthrone that seems to participate in the medicinal effects of this species. The aim of this work was to obtain an efficient micropropagation system of H. perforatum and to compare the hypericin content between in vitro and field-grown plants. Cultures were initiated from nodal segments of mature plants inoculated onto MS medium supplemented with 4.5 muM BA, kinetin, thidiazuron, individually or in combination with 0.05 muM NAA. Organogenic explants were observed on medium with either BA or kinetin alone or in combination of these with NAA. Subculture of organogenic explants onto the proliferation medium containing 4.5 muM BA promoted the organogenic response. The highest average of shoot production (52.6 shoots) was obtained on those explants induced in the presence of BA and NAA. Rooted plantlets were successfully acclimated. Analysis of hypericin contents showed that levels found in callus represented only 0.11 % of what was detected in adult plants, while shoots and leaves from in vitro plants showed similar hypericin levels to those found in the leaves of the field-grown plants, suggesting that the accumulation of this compound is related to leaf differentiation.

Clonal micropropagation of Crataeva adansonii (DC.) Prodr.: A multipurpose tree

A method of micropropagation through multiple shoot formation from axillary buds of mature tree and rootstock growths of Crataeva adansonii (DC.) Prodr. (a multipurpose tree) has been developed. Factors affecting multiplication rate included season, age of explant source, explant type, type of bud, position of bud on the foliage twig, type of medium, various additives, and explant implantation on the medium. The maximum number of buds was produced from the sixth to 10th axillary buds taken from foliage twigs of 40–50-d-old rootstock growth in the months of October to December. At this time of the year the contamination was minimum. Optimum response was recorded on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 3 mgl−1, 13.3μM) and 1-naphthaleneacetic acid (NAA; 0.05 mgl−1, 0.27 μM) after 21 d of culture. Shoot buds were further multiplied and maintained on BA (1–1.5 mgl−1, 4.4–6.7 μM) while shoots elongated on BA (0.1–0.5 mgl−1, 0.44–2.2 μM) supplemented medium. The number of shoots was further multiplied by using nodal segments of in vitro-regenerated shoots as microcuttings and repeated subculturing of stumps after excising the microshoots. In vitro rooting on growth regulator-free MS medium was possible with 70% of microshoots after 4 wk. From one nodal segment 150 plantlets were produced within 14 wk.

Micropropagation of calla lily (Zantedeschia albomaculata) via in vitro shoot tip proliferation

A method for the micropropagation of Zantedeschia albomaculata is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. Of the four cytokinins tested, 6-benzyladenine (BA) and thidiazuron (TDZ) were found to be more effective. An optimal concentration of BA (8.87 μM) or TDZ (4.54 μM) developed an average of 3.8 and 3.2 shoots per explant, respectively, but increasing concentrations of cytokinins often led to lower proliferation rate and stunted growth. Addition of auxins to the MS medium supplemented with 8.87 μM BA slightly enhanced multiple shoot formation in the explants. Multiplication of six cultivars of Zantedeschia genus comprising different flower types and colors were tested and achieved using only one regeneration medium (MS+8.87 μMBA+2.46 μM IBA). Different MS medium strength, air temperature (15, 20, 25, and 30°C) and light quality [fluorescent, red + blue, red and blue light provided by a LED (light-emitting diode) system] were used (without phytohormone) with the aim of stimulating in vitro shoot and root development. Half-strength MS or MS and cultures maintained at 25°C were found to be equally suitable for shoot tip culture of Z. albomaculata. Shoot elongation as well as fresh and dry weight were significantly increased when cultures were kept under red or blue light.

Provenance- and subculture-dependent variation during micropropagation of Gmelina arborea

Axillary shoot elongation, formation of multiple shoots and rooting of shoots were compared in nodal segment cultures of Gmelina arborea Roxb. from seedlings obtained from six provenances, over several subcultures. Provenance-dependent variation was observed with respect to these parameters. In addition, a subculture-dependent decrease was observed in multiple shoot formation and root induction. Seventy percent of the rooted plantlets were successfully hardened and transferred to soil. A transient decrease in photochemical efficiency was observed during the early stages of hardening, whereas ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) levels increased gradually as the plants acclimatized to photoautotrophic growth.

Changes in phenol content and peroxidase activity during in vitro organogenesis in Xanthosoma sagittifolium L.

Direct plant regeneration, multiple shoot formation and callogenesis were induced from cocoyam shoot tips cultured in vitro. At different stages of culture, phenol content, peroxidase activity and acidic soluble isoperoxidase patterns were analysed in plantlets. Results showed that phenol content of plantlets cultured on auxin-free media decreased with time, while it increased in those cultured on media supplemented with an auxin. Each form of morphogenesis induced with a growth regulator was preceded by an increase in total peroxidase activity. On hormone-free medium, organogenesis occurred (single shoot development and rhizogenesis), but there was no increase in total peroxidase activity. The appearance of isoperoxidase A2 was associated with root initiation, while the disappearance of isoperoxidase A5 and the appearance of isoperoxidase A6 preceded multiple shoot formation. These results indicate that total peroxidase activity was not a proper marker for organogenesis in cocoyam. Each form of morphogenetic differentiation is associated with an alteration of the acidic isoperoxidase pattern. These enzymes can be used as biochemical markers for rooting and multiple shoot initiation in cocoyam.

Somatic embryogenesis and organogenesis of Agave victoriae-reginae: Considerations for its conservation

Agave victoriae-reginae somatic embryos were produced through a callus phase from seedling stem segments cultured on MS medium. The optimal treatment was MS medium with 2.26 μM 2,4-D. Multiple shoot regeneration was induced from axillary buds from stem segments cultured on MS medium with 2.2–4.4 μM BA. Effect of MS and modified MS medium with 50% macronutrient concentration, both containing 2.2 μM BA and sucrose at the following concentrations, 20, 30, 45 and 60 g l−1, resulted in inconsistent multiple shoot formation. Shoots and somatic embryos formed by this indirect pathway could have a multicellular origin, which might lead to genetic variation. The direct development of pre-existent buds occurred on MS basal medium and increased in the presence of BA; this might be a pathway for the rescue of genotypes of endangered species. Embryos and shoots developed and grew roots on MS medium. Complete plantlets were obtained on MS basal medium. A total of 92% per cent of the plantlets survived and grew when transferred to the greenhouse. Agave micropropagation could supply the commercial plant demand, diminishing the gathering of seeds and plants of this endangered species from the wild.

Multiple shoot formation and plant regeneration from stem nodal explants of Paphiopedilum orchids

Stem nodal explants of Paphiopedilum philippinense hybrids (hybrid PH59 and PH60) directly formed shoots when cultured on a modified half-strength Murashige and Skoog (1962) basal medium supplemented with a combination of 2,4-dichlorophenoxyacetic acid (2,4-D: 4.52 and 45.25 μM) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 0.45 and 4.54 μM) for 6 mo. On hormone-free basal medium, the percentages of explants with shoots were 33.3% and 0% and the shoot numbers per explant were 1 and 0 in hybrid PH59 and hybrid PH60, respectively. In hybrid PH59, 4.52 μM 2,4-D plus 0.45μM TDZ induced a higher percentage of explants with shoots and shoot number per explant than did the hormone-free treatment. In hybrid PH60, although 4.52 μM 2,4-D and 0.45 μM TDZ promoted shoot formation, the highest shoot number was found with 4.52 μM 2,4-D alone. Plantlets, each having several roots, were obtained from regenerated shoots after transferring onto hormone-free basal medium for 3 mo. The plantlets were potted in sphagnum moss, and acclimatized well in a greenhouse.

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet ( Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N6 medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.