Shoot Multiplication Rate Research Articles

Shoot Bud Proliferation from Axillary Nodes and Leaf Sections of Non-toxic Jatropha curcas L.

Protocols for in vitro propagation of non-toxic variety of J. curcas through axillary bud proliferation and direct adventitious shoot bud regeneration from leaf segments have been established. Shoot bud proliferation from axillaries was assessed on an initial basal Murashige and Skoog (MS) salt medium supplemented with different concentrations of benzyladenine (BA), kinetin and thidiazuron (TDZ) followed by subculture to medium with 4.4-8.9 μM BA. Regardless of the concentration of BA in the subculture medium, shoot multiplication rate was optimum (10–12.3) with primary culture on medium supplemented with 2.3–4.5 μM TDZ. Efficient adventitious shoot regeneration from leaf tissues was achieved with culture on medium with 8.9–44.4 μM BA + 4.9 μM indole-3-butyric acid (IBA) followed by transfer to medium supplemented with 8.9 μM BA + 2.5 μM IBA. Similarity index between toxic Indian variety and the non-toxic variety based on 435 RAPD markers was 96.3%. Crossing studies followed by phorbol ester quantitation revealed that outcrosses with toxic J. curcas do not affect the phorbol ester content of seeds borne on the non-toxic variety.

Micropropagation of Clitoria ternatea Linn. (Fabaceae)— An important medicinal plant

An efficient protocol was established for in vitro shoot multiplication from nodal explants of Clitoria ternatea on semisolid Murashige and Skoog (MS) basal medium supplemented with 8.9μM 6-benzylaminopurine (BA). Inclusion of 1-naphthaleneacetic acid (NAA) in the culture medium along with BA promoted higher rates of shoot multiplication than BA alone. The rate of shoot multiplication was maximum (5.21) after 4 wk of culture on MS basal medium supplemented with 8.9μM BA and 1.34μM NAA. The elongated shoots rooted within 7–8d in half-strength MS basal salts supplemented with 1.34μM NAA and 2% (w/v) sucrose. About 85% of the rooted plantlets were acclimatized and transferred to the greenhouse.

In vitro regeneration and plant establishment of Tylophora indica (Burm. F.) Merrill: Petiole callus culture

Abstract A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid +2.5 μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5 μM TDZ. Individual elongated shoots were rooted best on half-strength MS medium containing 0.5 μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150 μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a gr...

An efficient in vitro method for mass propagation of Tylophora indica

A protocol of high frequency shoot organogenesis and plant establishment from stem derived callus has been developed for Tylophora indica (Burm. f.) Merrill. - an endangered medicinal plant. Callus was developed on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (80 %) of shoot multiplication was achieved on MS medium containing 5.0 μM kinetin. The developed shoots rooted best on half-strength MS medium supplemented with 0.5 μM indole-3-butyric acid (IBA). The in vitro raised plantlets with well developed shoot and roots were acclimatized successfully and grown in greenhouse.

In vitro micropropagation of endangered Rhododendron ponticum L. subsp. baeticum (Boissier & Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Anderson’s modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).

Cloning of Rauvolfia serpentina-An Endangered Medicinal Plant

Rauvolfia serpentina Benth. ex Kurz called sarpagandha(the snake root) in Hindi, belongs to family Apocynaceae. This plant is listed in earliest Ayurvedic medicinal text the Charaka Samhita(c. 700 B.C.) and has been used since at least that time to treat mental illness and insomnia. The roots of R. serpentina contain numerous alkaloids. Its indiscriminate use and poor method of conventional propagation have led this species to be included in the list of endangered plants. Thus, a need arises to generate an efficient protocol for cloning of R. serpentina in order to regenerate propagules to replenish depleting forests and meeting the demand of commercial cultivation. We report here on development of an efficient and simple process for large-scale clonal micropropagation of selected plants of R. serpentina and their successful field establishment. Nodal shoot explants produced multiple shoots (3-5 shoots per node) by axillary bud proliferation on Murashige and Skoog (MS) medium + 10 μM 6-benzylaminopurine (BAP) and 0.5 μM indole-3-acetic acid (IAA). Shoots were further multiplied on MS medium + 5.0 μM BAP + 0.5 μM IAA. After 3-4 subculture cycles (each cycle of 15-20 days) shoot multiplication rate increased up to 20-25 fold. Subsequently, shoots were multiplied on MS + 4.0 μM BAP to prevent hyperhydration. The micropropagated shoots were pulse-treated with 50 μM each of indole-3-butyric acid and P-naphthoxyacetic acid. About ninety-eight percent of in vitro- produced shoots rooted in vivo. The plantlets were transferred to bottles containing soilrite moistened with half-strength MS macrosalts. Eighty percent of these were hardened. Hardened plants were transferred to soils in black polybags. Micropropagated Rauvolfia serpentina plants grew normal in a nursery and these flowered, having set fruit/seed. The protocol developed is reproducible and can be used for germ plasm conservation and mass propagation of selected plant/ clone.

Factors affecting tissue culture of Damask rose ( Rosa damascena Mill.)

The present study was conducted to evaluate the regeneration ability of Damask rose. Single-node explants were surface sterilised with 10% chlorox for 15 min and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N 6-benzyladenine (BA) or kinetin (Kin) separately or in combination with different concentrations of indole-3-butyric acid (IBA). Combination of 2.5–3 mg/l BA and 0.1 mg/l IBA was the most suitable treatment for proliferation. In vitro derived shoots were subcultured four times on the fresh medium within a 4-week period. Other treatments such as explant orientation (horizontal, vertical and oblique) and explant wounding were also examined but did not affect shoot multiplication rate significantly. Several experiments were carried out to stimulate in vitro rooting of Damask rose. Application of different media such as MS, 1/2 MS, 1/3 MS and 1/4 MS with different concentrations of indole-3-acetic acid (IAA), IBA and naphthaleneacetic acid (NAA) did not produce satisfactory results. Quick-dip method using sterilised 0–2000 mg/l IAA, IBA and NAA solutions was also studied. Other treatments such as using various concentrations of abscisic acid (ABA) in combination with various concentrations of IAA, IBA and NAA, and using various concentrations of sucrose and agar did not produce any roots. The best treatment for rooting of shoots was 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 2 weeks in MS medium and then transferring the explants to MS medium without any growth regulator. Plantlets were acclimatised in a soil mixture consisting of peat moss and sand 1:1 (v/v) and successfully transferred to the greenhouse after 3 weeks.

Effect of triacontanol on the micropropagation of Costus speciosus (Koen.) SM. Using rhizome thin sections

The highest percentage of shoot regeneration of Costus speciosus was achieved using thin rhizome sections and triacontanol (TRIA). Factors affecting the rate of shoot multiplication and rooting with TRIA have been investigated. Initiation of shoot buds was observed when rhizome thin sections were cultured on B5 basal medium supplemented with 5μgl−1 TRIA. Shoots with two to three leaves produced roots when cultured on B5 basad medium supplemented with 2 μgl−1 TRIA. The well-rooted shoots were hardened and transferred to soil where they showed normal growth and a 100% survival rate was achieved. Results of this study showed that TRIA can be used as an effective growth regulator in the micropropagation and conservation of C. speciosus.

Shoot multiplication in Rauvolfia tetraphylla L. using thidiazuron

The effect of thidiazuron (TDZ) was investigated on in vitro shoot proliferation from nodal explants of Rauvolfia tetraphylla. Murashige and Skoog (MS) medium containing TDZ (0.5–10μM) was effective in inducing shoot buds and maintaining high rates of shoot multiplication on hormone free medium. The highest shoot regeneration frequency (90%) and mean number (18.50 ± 1.25) of shoots per explant were achieved from nodal segments cultured on MS medium supplemented with 5μM TDZ for 4 weeks prior to transfer to MS medium without TDZ for 8 weeks. The regenerated shoots rooted best on MS medium containing 0.5μM indole-3-butyric acid (IBA). Micropropagated plantlets were hardened to survive ex vitro conditions and were then established into soil.

In vitro regeneration of the medicinal woody plant Phellodendron amurense Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm2 sections of about 10-day-old leaves on MS medium enriched with 4.4 μM BAP and 1.0 μM NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 μM TDZ and 4.0 μM 2,4-D or 4.0 μM NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 μM BAP and 1.0 μM NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 μM IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.

In VitroCulture of Lowbush Blueberry (Vaccinium angustifoliumAit.)

SUMMARY Cultures of three lowbush blueberry (Vaccinium angustifolium Ait.) clones collected from natural stands in Newfoundland were established in vitro on a modified cranberry (V. macrocarpon Ait.) tissue culture medium containing zeatin (5 μM) or N6-[2-isopentenyl]adenine (2iP) (10 μM). Shoot proliferation with respect to shoot number per explant differed among clones at various concentrations of zeatin over two culture periods. Best total shoot proliferation was obtained when basal nodal segments were cultured in the medium supplemented with 2-4 μM zeatin. In another experiment, nodal explants were more productive than shoot tips. Shoots growing for more than 12 weeks on media that contained more than 4 μM zeatin occasionally produced adventitious shoot masses, which appeared to arise from dense calli growing at the base of the shoots in the medium. The lower concentration of sucrose and lower irradiance improved shoot proliferation with respect to vigor compared to the control treatments (30 g L_1 and 30 μmol m∼2 s_1, respectively). In all experiments with subculture, there was an increase in shoot multiplication rate for all clones. A 50-100-fold multiplication rate was obtained every 3 months with the clones.

Micropropagation of mature wych elm (Ulmus glabra Huds.).

Explants of mature vigorous donor trees of wych elm ( Ulmus glabra Huds.) that had not been previously exposed to Dutch elm disease were investigated for the influence of phytohormones and media on shoot multiplication rates and organogenic capacity. The regenerates were micropropagated from cultures that originated from 15-year-old progeny of plus trees. Two plus trees aged over 70 years showed recalcitrant responses. Thidiazuron in combination with 6-benzylaminopurine (BAP) induced a significantly higher number of shoots per explant than the most optimal BAP treatment (5.88 vs. 3.05 shoots). Woody plant medium and Dubovský minimal medium had no significant effects on shoot formation and multiplication rates. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. Two experimental field plots with 3-year-old in vitro-propagated trees were established.

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.

Rapid Micropropagation of Five Cultivars of Mulberry

Multiple shoots were initiated from nodal and shoot tip explants collected from mature trees of Morus alba L. cultivars Chinese White, Kokuso-27 and Ichinose, and M. multicaulis Perr. cultivars Goshoerami and Rokokuyaso after 2 weeks of culture. Nodal explants were more responsive than shoot tip explants. Murashige and Skoog basal medium was found to be most suitable medium and 6-benzylaminopurine was the most effective cytokinin for shoot induction. Explants collected between April and September evoked better response than the explants collected between October and March. Shoots were multiplied by transferring nodal explants excised from in vitro raised shoots onto a medium containing cytokinins. Sucrose was the most suitable carbon source examined for shoot multiplication. An increase in shoot multiplication rate was noticed upto 4 – 5 subcultures. Nodal explants rooted on an auxin-supplemented medium. The acclimatized plants were successfully transplanted in the field.

FORMATION OF TULIP BULBS IN VITRO

The propagation of tulip (Tulipa gesneriana L.) in vitro fully depends on vegetative bulb formation. To determine the internal and external factors influencing bulb development, the in vitro shoots of two cultivars of tulip were used for experiments: 'Blue Parrot' - with high rate of shoot multiplication but difficult-to-form bulbs, and 'Prominence' - well multiplying and easily forming bulbs. Effects of exogenous abscisic acid (ABA), methyl jasmonate (MeJA) and gibberellin (GA 3 ) as well as environmental factors (light, darkness and cooling period) and the kind of gelling agent on bulb formation were studied. Contents of endogenous plant hormones for in vitro shoots were determined by ELISA. The content of ABA and trans-zeatin riboside (t-ZR) in 'Blue Parrot' was significantly higher than in the shoots of 'Prominence'. After cooling (4°C), the content of endogenous ABA in shoots of 'Blue Parrot' decreased, but it was still 70% higher than in the 'Prominence' shoots before the low temperature treatment. The level of endogenous cytokinins (t-ZR and IPA - isopentenyladenosine) in 'Blue Parrot' significantly increased after cooling. Bulb formation of this cultivar was negatively influenced by ABA application. The results suggest that the poor capacity for bulbing observed in 'Blue Parrot' may result from high concentration of endogenous ABA and t-ZR in the shoots of this cultivar. A significant improvement of bulb development was obtained by extending the cooling period up to 14 weeks, as well as by application of MeJA and activated charcoal into the medium.

TEMPERATE FRUIT PLANT PROPAGATION THROUGH TEMPORARY IMMERSION

So far many research activities have been reported on automation in micropropagation; many liquid media systems for tissue culture have been proposed in this regard. In this work the temporary immersion system (TIS) has been tested for in vitro propagation of strawberry, pear, apple, peach, cherry, plum, raspberry and papaya. Because the objectives of the experiments were to assay the media composition, to establish the immersion-period length and to define environmental parameters, a simple double bottle device, manually operated, was used. The temporary immersion of the explants was achieved filling the bottle containing the explants with the medium transferred through a silicon tube from the second bottle, in which an overpressure was created by a manual air pump. The effects on growth were compared to those of the stationary liquid and agarized cultures (control). The shoot multiplication rate was very high in strawberry, showing the typical juvenile unilobate morphology, raspberry and papaya. In pear the multiplication efficiency was strictly genotype dependent; TIS revealed to be highly efficient to prevent the apices necrosis. For the other species the TIS strongly affected the quality of the explants preventing the necrosis and the hyperhydricity. All the species maintained the juvenile habit during the TIS culture. Data on photosynthetic pigments and sugar contents seem to suggest a positive influence of TIS on the autotrophic activity.

MICROPROPAGATION OF JUVENILE EUCALYPTUS CITRIODORA

A protocol for in vitro propagation in Lemon eucalypt (Eucalyptus citriodora) has been developed. Shoot apices or medium or basal segments from aseptically grown seedlings were cultured on Murashige and Skoog (MS), Woody Plant Medium (WPM) and Schenk and Hildebrandi (SH) supplemented with BA at 0, 0.5, 1, 2 and 4 mg L -1 or Kin at 0, 0.5, 1.2 and 4 mg L -1 and NAA at 0.5 mg L -1 for multiplication and IBA at 0, 1, 2 and 3 mg L -1 for rooting. Highest shoot multiplication rates were obtained when the basal explants were cultured on MS medium supplemented with 0.5 mg L -1 BA. Regenerated shoots were rooted on 0.2 strength of MS medium supplemented with 1 or 2 mg L -1 IBA.

Clonal propagation, encapsulation and reintroduction of Ipsea malabarica (Reichb. f.) J. D. Hook., an endangered orchid

Rapid clonal propagation and encapsulation of in vitro-formed bulbs of Ipsea malabarica (Reichb. f.) J. D. Hook., an endemic and endangered orchid of the Western Ghats of Kerala, and its reintroduction to the natural habitat were accomplished. Rhizome segments of Ipsea cultured on half-strength Murashige and Skoog (MS) medium supplemented with 6.97 μM kinetin induced the highest number of shoots, at the rate of four shoots per explant within 50 d. Transfer of the isolated shoots increased the rate of shoot multiplication to more than 10 shoots. Subsequent culture enhanced the number of shoots. No decline of shoot multiplication was observed up to the 10th subculture. Shoots developed bulbs during culture which developed into rhizomes. Sucrose at 6–8% reduced the time for the development of bulbs and rhizomes. Roots were developed from the base of the developed shoots as well as from the bulbs. Isolation and culture of bulbs also developed 5–10 shoots within 50 d. Encapsulated in vitro-formed bulbs cultured either on hormone-free halfstrength MS or 6.97 μM kinetin-supplemented medium facilitated 100% conversion. As a step to conservation in situ, 50 plantlets were reintroduced into their natural habitat, i.e. at Vellarimala (at 1300 m height) of the Western Ghats of Kerala, and flowered normally. Development of more than 40 000 plantlets starting from a single explant is possible within 250 d. This threatened endemic orchid stands to benefit greatly from the established protocol and will hopefully curtail the threat of extinction.

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l−1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l−1 NAA+11% (w/v) sucrose) supplemented with 10 μM JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 μmol m−2 s−1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.

In vitro growth and shoot multiplication of Wrightia tomentosa Roem et Schult in a controlled carbon dioxide environment

In vitro growth and multiplication of shoots of a woody tree species Wrightia tomentosa in a controlled carbon dioxide environment was studied. The cultures were grown on BA supplemented MS medium with or without 3% sucrose. A range of CO2 concentrations (0.0, 0.6, 10.0 and 40.0 g m−3) was controlled in small chambers by using solutions of NaHCO3, Na2CO3, KHCO3 and K2CO3. To obtain a CO2-free environment, a saturated solution of KOH was kept in the chambers. It was concluded that the growing shoot cultures required either sucrose in the medium as a carbon source or an ambient CO2 environment. Complete absence of a carbon source caused severe browning of the shoots and death within 30 days. The cultures grew better with 10.0 g m−3 carbon dioxide in the environment than with 3.0% sucrose in the medium. With both CO2 and sucrose being available, the best response was obtained at 0.6 g m−3 CO2 in the chamber. At this concentration the rate of shoot multiplication was nearly double the standard rate obtained when exposed to the natural CO2 level and sucrose-supplemented medium. Total fresh and dry weight, leaf number and area per cluster also showed the best response under this condition.