High frequency in vitro propagation of Isodon wightii (Bentham) H. Hara

A protocol for in vitro propagation of Isodon wightii (Bentham) H. Hara from nodal segments was developed. Multiple shoots were successfully established on half strength MS medium supplemented with 4.4 μM BA. Enhancement of shoot multiplication and elongation was achieved on half strength MS medium supplemented with 4.4 μM BA and 1.4 μM GA₃. The regenerated shoots were rooted successfully on half strength MS medium supplemented with 4.9 μM IBA. Acclimatization of in vitro rooted shoots was successful. The in vitro regenerated plants grew well in the greenhouse without any phenotypic changes.     

In vitro plantlet regeneration of<Emphasis Type="Italic"> Schinopsis balansae</Emphasis> (Anacardiaceae)

A protocol for the in vitro regeneration of rooted plants from nodal single bud segments of 10-year-old Schinopsis balansae trees was developed. Nodal segments were harvested from actively growing shoots of plants grown from seeds and maintained in pots under greenhouse conditions, and from epicormic shoots obtained by forced flushing of branches. Culture of nodal segments on nutrient medium containing the mineral salts and vitamins of Murashige and Skoog medium at 1/4 strength (1/4 MS), supplemented with 100 mg l^–1 ascorbic acid, 3% sucrose, and 5–15 M 6-benzyladenine resulted in regeneration of multiple shoots. Rooting of regenerated shoots was observed in 1/4 MS medium with vermiculite as the substrate and supplemented with 7.5 M indolbutyric acid.     

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

Padar (Stereospermum personatum, family Bignoniaceae) is a well-known medicinal tree. Its complete regeneration occurred through shoot bud culture in vitro. The seeds germinated sequentially on plastic trays and polyethylene bags for 21 days served as explants source. Nodal segments from the seedlings were established on MS medium supplemented with 4.44 μM BA, in which 86.6% nodes showed shoot bud elongation. Then, nodal segments from the developed shoots were cultured on MS medium with several BA concentrations; best shoot multiplication was obtained with 0.44 μM BA. In a second experiment where PVP was added to proliferation medium, nodal segments from developed shoots produced maximum 2.78 shoots per node. The nodal segments showed shoot multiplication up to seventh subculture on. Finally, shoots were rooted on MS medium with 2.46 μM IBA. The plants transferred to net pots containing coco-peat were acclimatized in green house, where more than 80% plants survived and grew normally.     

Micropropagation of <Emphasis Type="Italic">Pueraria tuberosa</Emphasis> (Roxb. Ex Willd.) and determination of puerarin content in different tissues

Pueraria tuberosa, a medicinally important leguminous plant, yielding various isoflavanones including puerarin, is threatened, thus requiring conservation. In this study, fresh shoot sprouts of P. tuberosa, produced by tubers, were used as explants for in vitro micropropagation. Surface-sterilized nodal shoots were incubated on Murashige and Skoog (MS) medium supplemented with 8.88 μM benzyladenine (BA), 50 mg l⁻¹ ascorbic acid, and 25 mg l⁻¹ of each of citric acid and adenine sulphate. Cut ends of nodal stem segments rapidly turned brown, and cultures failed to establish. When 100 mg l⁻¹ ascorbic acid (ABA) and 25.0 mg l⁻¹ polyvinyl pyrrolidone (PVP) were added to the medium, explants remained healthy, and cultures were established. Bud-breaking of nodal stem explants resulted in multiple shoot formation. Shoots proliferated (35–40 shoots per culture vessel) on MS medium as described above, but supplemented with 4.44 μM BA and 0.57 μM indole acetic acid (IAA) and additives. After 4–5 passages, proliferating shoots exhibited tip-browning and decline in growth and multiplication. However, when shoots were transferred to fresh shoot proliferation medium supplemented with 2.32 μM kinetin (Kn), sustained growth and high rate of shoot proliferation (50–60 shoots per culture vessel) was observed. Shoots rooted when transferred to medium consisting of half- strength MS medium with 9.84 μM indole butyric acid (IBA) and 0.02% activated charcoal. Alternatively, individual shoots were pulsed with 984.0 μM IBA and transferred to glass bottles containing sterile and moistened soilrite. These shoots rooted ex-vitro and were acclimatized in the greenhouse. Plants were then analyzed for puerarin content using HPLC, and leaves showed maximum accumulation of purerarin.     

Alginate-encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.)

Nodal segments obtained from in vitro proliferated shoots of Eclipta alba (L.) Hassk, were encapsulated in calcium alginate beads for large-scale clonal propagation, short-term conservation and germplasm exchange and distribution. The best gel complexation was achieved using 3% sodium alginate and 100 mM CaCl₂·2H₂O. Maximum percent response (100%) for conversion of encapsulated nodal segments into plantlets was obtained on 0.7% agar-solidified full-strength MS medium containing 0.88 μM BAP. Encapsulated nodal segments could be stored at low temperature (4°C) up to 60 days with a survival frequency of 51.2%. The well-developed plantlets regenerated from encapsulated nodal segments were hardened-off successfully with 90% survival frequency.     

Effect of plant growth regulators on plant regeneration of Dioscorea remotiflora (Kunth) through nodal explants

Dioscorea remotiflora (Kunth) is an important wild plant that produces tuberous roots used as a source of food in the Western part of Mexico. Lack of planting material and inefficiency of traditional methods of propagation are the main constraints for implementing large-scale cultivation. In contrast, tissue culture techniques allow increasing multiplication and rapid production of plant material. In this regard, leaves or nodal segments were incubated on MS, B5 and WPM culture media with different PGRs in order to obtain an efficient micropropagation protocol. Leaves explants were unable to inducing shoots or callus. However, nodal segments produced axillary shoots and/or callus in all culture media. MS containing 2.33???M KIN was the most suitable to inducing shoots; an average of 6.6 shoots per segment for 100?% explants was obtained, which displayed also the greater number of nodes (5.0) and leaves (7.9) per segment. A decrease on shoot proliferation was observed combining BA or KIN with 2,4-D or NAA. However, small brownish callus were induced on 100?% of segments using 2.33???M KIN with 5.37???M 2,4-D or 9.30???M KIN plus 2.69???M NAA. In contrast, by adding 2.69???M NAA, 66.4?% of the nodal segments formed shoots and produced also yellowish friable callus on the base of the shoots. Shoots were easily rooted with 8.28???M IBA (96.9?%), displaying the greatest root and shoot biomass, but maximum number of tuberous roots, and root or tuberous root biomass was produced increasing IBA (20.7???M).     

Micropropagation of Paulownia fortuneii through in vitro axillary shoot proliferation

Primary cultures were established with nodal segments from juvenile shoots of two- year-old Paulownia fortuneii trees from a clonal plantation in Andhra Pradesh. A medium containing half-strength MS salts + RAP (1 mg/L) + sucrose (2%) produced optimum bud break in nodal explants. The same basal medium with reduced hormone level (0.5 mg/L) supported maximum multiplication of secondary cultures of P. fortuneii (1:6 in 6 weeks). Specific treatments were tested to enhance this rate of multiplication. In one approach, five to six week old in vitro grown shoots were ratooned (cutting the main shoot at the bottom leaving one node). The stumps (ratooned basal node) produced 2 to 3 axillary shoots, which grew into 4 to 5 nodes by 3 weeks; thus, providing additional shoots from the same explant. This provided 30% additional shoots in 4 cycles. Secondly, reducing the light intensity to 1200 lux resulted in higher shoot elongation, i.e, formation of 8 nodes in 5 weeks with healthier shoots than the normal intensity of 3000 lux under which only 6 nodes were produced in 6 weeks. In vitro-grown shoots could be successfully rooted ex vitro in vermiculite + cocopeat mixture (1:1 v/v) under 90% humidity, transferred to soil in polybags for hardening in the green house for 2 weeks and shifted to shade net for further hardening. After one month, the plants could be successfully transplanted to field with 95% survival. Micropropagated plants showed an excellent growth in the field attaining a height of 1.5 m and a collar diameter of 2.8 cm in 3 months.     

An improved micropropagation of Terminalia bellirica from nodal explants of mature tree

An efficient and improved in vitro propagation method has been developed for Terminalia bellirica, a medicinally important tree from nodal explants of 10-year-old mature tree. Shoot multiplication was influenced not only by cytokinin types, their concentrations and their interaction with auxin but also by successive transfer of mother explants for different passages, subculture of excised shoots on fresh medium and different medium composition. MS medium containing 2.22 μM BAP was found to be the best for shoot multiplication in a single step. After excision of newly formed shoots, mother explants successively transferred to the same medium produced maximum shoots per explant after IV passage. Further enhancement in morphogenetic response occurred when excised shoot clumps (2–3 shoots) were subcultured on MS medium supplemented with 2.22 μM BAP, 1.16 μM Kn and 0.57 μM IAA. Half-strength MS medium supplemented with 24.60 μM IBA and 100 mg l⁻¹ AC was most effective for rooting of the shoots. To reduce labor, cost and time, an experiment on ex vitro rooting was also carried out and it was observed that highest percent shoots rooted ex vitro when treated with 2,460 μM IBA for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. In comparison to plantlets developed from in vitro rooted, percent survival of plants those rooted ex vitro was significantly higher. Use of ex vitro rooting technique for plant production serves as a more economical option; therefore, present method can be used for large-scale commercial production of this medicinally important tree.     

The effect of polyamines on the efficiency of multiplication and rooting of <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal and content of some withanolides in obtained plants

An efficient mass multiplication protocol was developed for Withania somnifera (L.) Dunal from nodal explants of field-grown plants on Murashige and Skoog medium (MS) supplemented with 6-benzyladenine (BA) [1.5 mg L^−l], indole-3-acetic acid (IAA) [0.3 mg L^−l] and with the addition of polyamine, spermidine (20 mg L^−l) (shoot multiplication medium). A total of 46.4 shoots were obtained from nodal explants and they were elongated in the same medium in a culture duration of 6 weeks. The elongated shoots produced roots in MS medium fortified with putrescine (20 mg L^−l) after 4 weeks, and all the rooted plants were successfully hardened and acclimatized with a survival rate of 100%. An average of 276 shoots (46 × 6) was produced when at least six nodal explants obtained from each of the 46 in vitro grown shoots were cultured by microcutting method in the same shoot multiplication medium. On an average, 12,696 plants could be produced from all the shoots (276 × 46) by microcuttings in a period of 7 months. HPLC revealed a significant increase in the quantities of withanolide A, withanolide B, withaferin A and withanone in the leaves, stems, and roots of in vitro regenerated plants compared to the field-grown parent plants. Ploidy analysis using flow cytometry revealed genetic stability of in vitro regenerated plants. This protocol will be useful for scale-up production of withanolides on commercial scale.     

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N ⁶-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.     

A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry

An efficient system to regenerate shoots on excised leaves of greenhouse-grown wild lowbush blueberry (Vaccinium angustifolium Ait.) was developed in vitro. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, medial, and basal segments of the leaves was tested. Leaf cultures produced multiple buds and shoots with or without an intermediary callus phase on 2.3–4.5 μM TDZ within 6 wk of culture initiation. The greatest shoot regeneration came from young expanding basal leaf segments positioned with the adaxial side touching the culture medium and maintained for 2 wk in darkness. Callus development and shoot regeneration depended not only on the polarity of the explants but also on the genotype of the clone that supplied the explant material. TDZ-initiated cultures were transferred to medium containing 2.3–4.6 μM zeatin and produced usable shoots after one additional subculture. Elongated shoots were dipped in 39.4 mM indole-3-butyric acid powder and planted on a peat:perlite soilless medium at a ratio of 3:2 (v/v), which yielded an 80–90% rooting efficiency. The plantlets were acclimatized and eventually established in the greenhouse with 75–85% survival.     

Plant regeneration from in vitro leaves of the peach rootstock ‘Nemaguard’ (<Emphasis Type="Italic">Prunus persica</Emphasis> × <Emphasis Type="Italic">P. davidiana</Emphasis>)

A complete protocol for adventitious shoot regeneration was developed from the leaves of peach rootstock ‘Nemaguard’(Prunus persica × P. davidiana) grown in vitro. Shoot explants were cultured in vitro in Murashige and Skoog medium supplemented with 3.55 μM 6-benzyladenine and 7.38 μM indole-3-butyric acid (IBA). Non-expanded leaves along with their petioles from 3-week-old in vitro-grown shoots were used as explants. Regeneration percentage was influenced by plant growth regulators, basal medium, explant type, dark period, and gelling agents. Optimal regeneration was observed with leaf explants wounded by transverse cuts twice along the midrib and first incubated with abaxial surfaces facing upward in the dark for 3 weeks, and then transferred to the light and cultured with the adaxial side in contact with regeneration medium, as seen on 1/2 MS, woody plant medium or Schenk and Hildebrandt medium supplemented with 9.08 μM thidiazuron, 0.54 μM IBA and 0.25% agar. This produced the highest regeneration percentage at 71.7% and a mean of 5.74 ± 3.24 shoots on 1/2 MS medium. Adventitious shoots were rooted (98.3–100%) and rooted plantlets survived after acclimatization to the greenhouse.     

Induction,growth and direct rooting of adventitious shoots of Begonia x hiemalis

The efficiency of commercial micropropagation programs for Begonia x hiemalis depends on the production of large adventitious shoots for easy handling and on effective rooting and acclimatization procedures. Maximum induction of adventitious buds on petiole segments occurred in response to NAA (0.1 mg, l^-1) and BA (0.5 mg l^-1), but continued shoot growth was limited. With a lower concentration of BA (0.1 mg l^-1) fewer shoots were produced but shoot growth was enhanced. With a combined agar/liquid culture program the low BA (0.1 mg l^-1) medium produced 50 percent more shoots larger than 1 cm than did the high BA (0.5 mg l^-1) medium. In vitro rooted explants developed weak root systems and acclimatization losses occurred during adaptation to greenhouse conditions. Adventitious shoots treated with commercial rooting powder and placed directly in mist frames produced much stronger root systems and could be adapted to greenhouse conditions without loss. The elimination of the in vitro rooting stage also simplifies the micropropagation program.Contribution No. 743     

Direct shoot regeneration from nodal,internodal and petiolar segments of <Emphasis Type="Italic">Piper longum</Emphasis> L. and in vitro conservation of indexed plantlets

Three explants namely, nodal, internodal and petiolar segments were used to establish in vitro cultures of Piper longum. Multiple shoots were induced on semi-solid Murashige and Skoog (MS) medium supplemented with 1 mg/l 6-benzyladenine (BA). Addition of ascorbic acid (40 mg/l) considerably reduced browning of tissue and medium. Best shoot regeneration was observed from petiolar explants and was, therefore, used for all further studies. An indexing method was introduced for checking bacterial contamination in well established shoot multiplication cultures. It was found that bacterial infection was quite high in shoots derived from nodal and internodal explants while it was least in those obtained from petiolar segments. Only shoots that indexed negative for endogenous bacteria were used for proliferation and in vitro conservation studies. At the end of 4 weeks in proliferation medium which consisted of MS supplemented with 0.5 mg/l BA and 40 mg/l ascorbic acid as many as 22 shoot buds of 41 mm length could be obtained. Shoot buds developed into clusters for ease of further proliferation. A step of shoot elongation for 2 weeks in liquid MS basal medium was found to be beneficial for getting long and healthy shoots for rooting. Single shoots were rooted in 0.25 mg/l indole butyric acid that could be successfully acclimatized under nethouse conditions. A conservation strategy was also developed. The shoot cultures could be maintained without subculturing for as long as 8 weeks in MS medium supplemented with 1 mg/l paclobutrazol (PBZ) and 40 mg/l ascorbic acid.     

In Vitro Plantlet Formation in Mangosteen (Garcinia mangostana L.)

Optimum conditions were determined for in vivo growth and multiplicationof Garcinia mangostana L. using explants from aseptically germinatedseedlings and field-grown plants. Proliferating shoots wereobtained from cotyledon segments cultured on modified Murashigeand Skoog's (1962) medium with 6-benzylaminopurine. Juvenileleaf segments produced adventitious buds on Woody Plant Medium(Lloyd and McCown, 1981). Root segments gave few buds. Shoottip, nodal, and internodal explants gave multiple axillary andadventitious buds. Shoots were multiplied by enhanced axillaryand adventitious bud formation. The shoots were rooted withindolebutyric acid treatment. Rooted shoots were readily establishedin vermiculite: sand (1:1) mixture. Garcinia mangostana L., Mangosteen, tissue culture, shoot regeneration, bud development     

Immunoadjuvant saponin production in seedlings and micropropagated plants of <Emphasis Type="Italic">Quillaja brasiliensis</Emphasis>

The leaves of Quillaja brasiliensis, a native tree species of southern Brazil, accumulate saponins which have adjuvant activity in vaccines. An efficient micropropagation system for Q. brasiliensis was developed. Plants were obtained from aseptically germinated seeds. High rates of germination were observed on filter paper and aseptic medium substrate (sucrose and light exposure did not affect the germination response). Stem nodal segments were inoculated into Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine. Each explant produced multiple shoots, which elongated satisfactorily on the same medium. Elongated shoots were rooted in a modified MS medium with or without auxins. Best rooting responses were observed in medium containing 10 mg l⁻¹ indole-3-acetic acid under continuous exposure. The rooted explants were acclimatized and successfully transferred to soil, yielding approximately 95% survival after 10 mo. Leaf content of immunoadjuvant saponins in micropropagated plants was not affected by auxin type used for rooting and was comparable to that of field-grown trees. The results indicate that Q. brasiliensis represents an alternative and readily renewable source of biomass for the production of bioactive saponins.     

In vitro micropropagation of Basella rubra L. through proliferation of axillary shoots

In vitro micropropagation protocol for Basella rubra regeneration was tried through proliferation of axillary shoots of the potted mature plant. The improved seed germination (70%) was recorded upon 2% urea treatment. The nodal shoot segments from matured potted plant were used to initiate the multiple shoot proliferation. The shoot segments exhibited 70% shoot initiation when cultured on Murashige and Skoog (MS) medium supplemented with Indole-3-acetic acid (IAA)?+?N₆ – Benzylaminopurine (BAP) (0.25?+?2.0 mg/L) and BAP?+?Kinetin (Kin) (2.0?+?0.5 mg/L) respectively. Multiple shoots (5–6) were obtained on MS medium supplemented with BAP?+?Kin and IAA?+?BAP respectively. When compared with silver nitrate (AgNO₃) (2–40 µM) and activated charcoal (AC) (0.1–1.0%), the MS medium devoid of any plant growth regulator showed good number of shoots (5.48?±?2.42), elongation (15.64?±?2.42 cm) and root length (14.52?±?2.78 cm). Upon transferring of regenerated microshoots to MS medium, simultaneous elongation of shoots with more shoot number, shoot length and rooting was achieved during four subcultures that carried out at 6 weeks’ interval. The regenerated in vitro shoots showed 100% rooting in MS medium and also in MS medium supplemented with 0.1–1.0% AC. Hundred percent survival of micropropagated shoots well rooted was established successfully under greenhouse condition and the plants were subsequently acclimatized and transferred to the field conditions wherein 90% success rate was noted.

     

From leaf explants to hanging rooted plantlets in a mist reactor

A mist bioreactor using a disposable bag as a culture chamber was used to propagate single nodal explants of Artemisia annua into rooted plantlets that were ready for soil using a single batch (one-step) culture. To vertically scale plant growth inside the mist reactor, poly-l-lysine (PLL)-coated 70 µm nylon mesh and solid polypropylene sheeting were used for explant attachment. Both manually chopped and blender-chopped (blenderized) shoot tissues were attached to PLL-coated substrates. Compared to blenderized shoots, manually chopped tissues were larger with better attachment to PLL-coated substrates. Regardless of substrates or explant preparation method, 80–95 % of initially attached shoot tissues remained attached to PLL-coated surfaces after being misted with culture medium for 24 h. New shoot proliferation increased about tenfold as the size of blenderized shoot tissue increased. To reduce callusing during shoot proliferation and thus stimulate root initiation, original shooting medium was reduced to half strength of phytohormone and Murashige and Skoog salts. The duration of shoot proliferation was also reduced from 2 to 1 week. Original rooting medium was then further improved with NAA or IBA. After successive shooting, rooting and in vitro acclimatization, the nodal explants attached to PLL-coated hanging strips and developed into fully rooted plantlets in the mist reactor. Although most of the large rooted plantlets detached from the hanging strips by the time of harvest, they had fully functional stomata and were later successfully established in the soil, suggesting this “hanging garden” technology may prove useful for micropropagation.

     

In vitro rapid multiplication and propagation of <Emphasis Type="Italic">Cardiospermum</Emphasis><Emphasis Type="Italic">halicacabum</Emphasis> L. through axillary bud culture

A simple, rapid and efficient protocol for micropropagation of Cardiospermum halicacabum via axillary bud multiplication has been successfully developed. The organogenic competence of nodal segments was investigated on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyladenine (BA), kinetin (Kn), thidiazuron (TDZ) and 2-isopentenyladenine (2-iP). Multiple shoots differentiated directly without callus mediation within 4 weeks when explants were cultured on a medium fortified with cytokinins. The maximum number of shoots (14.83 ± 0.52) was developed on a medium supplemented with 0.3 μM TDZ. Such proliferating shoots when subcultured onto MS media devoid of TDZ gave the highest rate of shoot multiplication (35.66 ± 1.00) by the end of fourth subculture passage. Elongated shoots were rooted on 1/3 MS medium augmented with 0.5 μM IAA. The plantlets thus obtained were successfully hardened and transferred to greenhouse.     

Thidiazuron-induced shoot organogenesis and production of hepatoprotective lignan phyllanthin and hypophyllanthin in <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Callus cultures from nodal and leaf explants of Phyllanthus amarus were established on Murashige and Skoog (MS) medium with various combinations of auxins and cytokinins. The leaf-derived callus induced on 2.26 μM 2,4-dichlorophenoxyacetic acid (2, 4-D) + 2.32 μM Kinetin (Kin) upon transfer to medium containing thidiazuron (TDZ) exhibited higher shoot regeneration (32.4 ± 1.3 shoots per culture). Four-week-old shoots rooted readily on 1.5 μM Indol acetic acid (IAA)-containing medium and were successfully acclimatized with 98% survival. The lignans, Phyllanthin (PH) and Hypohyllanthin (HPH), of leaf extracts from naturally grown plants were identified by using TLC, HPLC and H1-NMR. The PH and HPH production in the regenerated shoots was compared to their production in callus cultures, plants under field conditions and in naturally grown plants. The regenerated shoots on MS + 2.27 μM TDZ produced about two times higher PH and HPH than the leaves of naturally grown plant. The present study provides a useful system for further studies on in vitro morphogenesis, elicitor-assisted production of PH and HPH and A. rhizogenes-mediated genetic transformation in Phyllanthus amarus.