Rapid Mass Propagation Research Articles

Establishment of an efficient in vitro regeneration protocol for rapid and mass propagation of Dendrobium chrysotoxum Lindl. using seed culture.

An efficient in vitro regeneration protocol from seed culture has been established successfully for Dendrobium chrysotoxum, an epiphytic orchid having tremendous ornamental and medicinal values. Seed germination response was encouraging in Mitra (M) medium enriched with different combinations of auxins and cytokinins. Medium supplemented with 0.4% activated charcoal (AC), 2 mg/L 6-benzyl amino purine (BAP), and 2 mg/L indole-3-acetic acid (IAA) produced best seed germination percentage in 2 weeks of culture. Incorporation of higher concentration of kinetin (KN) or BAP in combination with low auxin in medium induced pronounced shooting and leaf formation. Reduction in leaf development was evident when cytokinins exist singly in medium indicating synergistic effect of auxin and cytokinin in leaf induction. Presence of elevated level of indole-3-butyric acid (IBA) or 1-naphthalene acetic acid (NAA) with low cytokinin content in medium generated more in vitro rooting, though IBA was found to be more effective in rooting induction as compared to NAA. The in vitro protocol for asymbiotic seed germination developed from the present investigation can be used for rapid mass propagation of this highly important Dendrobium orchid species.

Non-Destructive Assessment of Growth Performance of Embryogenic Suspension Culture of Pinus kesiya (Royle ex. Gord.) in Shake-Flask and Self-Designed Bubble Bioreactor and Successful Regeneration of Plantlets from the Culture Systems

Embryogenic suspensions of Pinus kesiya (Khasi pine) derived from embryogenic cultures obtained from immature zygotic embryos (intact megagametophytes) of Pk-04 genotype were grown in shake-flasks and self-designed bubble bioreactors. Conifer somatic embryogenesis could find its utility for rapid mass propagation if grown as suspension cultures. To minimize the destruction of embryogenic cultures during study, a few growth characteristics viz., sedimented cell volume and number of stage-I (early stage) and -III (cotyledonary) embryo formation in suspensions were determined over a 21-day period. Compared to shake-flasks, proliferation in bioreactors resulted in increased biomass. The suspension culture showed a mixture of freely suspended as well as aggregates of stage-I (early stage) embryos. Maturation of somatic embryos was done on gelled medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage-III (cotyledonary) embryos formed in bubble bioreactor were significantly higher than shake-flask cultures with both solid medium and submerged cultures. The cotyledonary embryos germinated successfully producing plantlets on germination medium.

NFT 수경재배에서 양액 종류 및 삽수 크기가 고구마 바이러스 무병주 생육에 미치는 영향

To develop a technique for mass-propagation of virus-free sweet potato [Ipomoea batatas (L.) Lam.] plant using nutrient film technique (NFT), the growth characteristics of 4 cultivars as affected by nutrient solution composition and cutting size were investigated. 72 cells (35 mL/cell) plug trays filled with vermiculite and perlite (1:1, v/v) were used. Vine length, fresh and dry weights of virus-free plants were the greatest in the nutrient solution recommended by National Horticultural Research Station in Japan, followed by that recommended by National Institute of Horticultural & Herbal Science in Korea, and Yamazaki's nutrient solution for lettuce. The growth of uppershoot cuttings was the best among 4 subsections of cutting. Vine length, and fresh and dry weights increased in the longer cutting treatments, and were better in 'Shinzami' and 'Yeonhwangmi' than those in 'Mannami' and 'Shincheonmi'. Vine diameter and length of the longest root were not significantly affected by the cutting size and cutting source. The growth characteristics of the single node cutting were not significantly different from those in 2-node cutting. The efficiency of rapid mass-propagation could be promoted with single node cuttings and uppershoot cuttings grown in NFT system.

In vitro morphogenic response and metal accumulation in Albizia lebbeck (L.) cultures grown under metal stress

An efficient regeneration protocol for rapid mass propagation and uptake of heavy metals in Albizia lebbeck (L.), a fast growing, medicinally as well as economically important timber yielding tree was developed. Nodal segments derived from a 20-year-old tree were cultured on MS (Murashige and Skoog) medium supplemented with 10 μM 6-Benzyladenine (BA) and 1 μM α-Naphthalene acetic acid (NAA) showed optimum shoot regeneration frequency (76.6%), number of shoots (23.2 ± 0.28) per explant and shoot length (2.86 ± 0.08 cm) after 10 weeks of culture. After standardizing a reliable protocol for micropropagation, effects of ZnSO4 (0.06–0.48 mM), CuSO4 (0.02–0.2 mM) and CdCl2 (0.0001–0.001 mM) on shoot morphogenesis were also assessed. The regenerated shoots maintained on maintenance medium (MS + 10.0 μM BA + 1.0 μM NAA) containing ZnSO4 (0.06 mM) showed maximum response in terms of shoot number (24.5 ± 0.83) and length (5.9 ± 0.05 cm) after 10 weeks of culture. Proline content showed an increasing trend while chlorophyll (a and b) content exhibited decreasing trend with an increased metal concentrations compared to MM cultures, and maximum increase in proline and decrease in chlorophyll content was recorded in cultures grown on Cd-enriched medium. Best rooting was accomplished on half strength MS medium with 2.0 μM IBA and ZnSO4 (0.06 mM). The plantlets thus obtained were successfully hardened and transferred to greenhouse with 75% survival rate and exhibited normal morphological characteristics compared to donor plant.

Production of psoralen by in vitro regenerated plants from callus cultures of Psoralea corylifolia L.

Psoralea corylifolia is an attractive, endangered annual producing various bioactive compounds of medical importance. This plant contributes to Indian pharmaceutical and cosmetic industries for the production of commercial medicines, Ayurvedic skin care ointments and soap. The influence of various plant growth regulators (PGRs) and additives on high-frequency rapid adventitious shoot regeneration from transverse thin cell layer (tTCL) hypocotyl explants of P. corylifolia was investigated. Organogenic callus was obtained in tTCL hypocotyl explants on Murashige and Skoog (1962) medium supplemented with 15 μM naphthaleneacetic acid (NAA) and 3 μM benzylaminopurine (BA). The highest adventitious shoot regeneration (107.5 shoots per explant) was achieved in culture when transferred to half-strength solid MS medium. The regenerated shoots were rooted and the plantlets successfully acclimatized in moistened (1/8-MS basal salt solution with 3 μM indole-3-butyric acid (IBA), 1 μM 2-isopentenyladenine (2iP) and 100 mg l−1 Bavistin (BVN)); garden soil, farmyard soil and sand (2:1:1, v/v/v). The acclimatized plants produced flowers in the growth chamber. When planted in the field these plants set viable seed. The psoralen content in different tissues of ex vitro and naturally-grown plants was determined by high-performance liquid chromatography (HPLC). The highest psoralen content was recorded in seeds from naturally-grown (6.48 μg g−1 DW) and ex vitro plants (6.46 μg g−1 DW). This system can be used for rapid mass propagation of P. corylifolia, for conservation strategies, and to produce phytomedicines.

Shoot multiplication kinetics and hyperhydric status of regenerated shoots of gladiolus in agar-solidified and matrix-supported liquid cultures

In vitro shoot regeneration of gladiolus in three different culture systems, viz., semi-solid agar (AS), membrane raft (MR), and duroplast foam liquid (DF) cultures was evaluated following the kinetics of shoot multiplication and hyperhydricity at optimized growth regulator combinations. Compared to the AS system, matrix-supported liquid cultures enhanced shoot multiplication. The peak of shoot multiplication rate was attained at 18 days of incubation in the MR and DF systems, whereas the maximum rate in the AS system was attained at 21 days. An early decline in acceleration trend was observed in liquid cultures than the AS culture. The hyperhydric status of the regenerated shoots in the different culture systems was assessed in terms of stomatal attributes and antioxidative status. Stomatal behavior appeared to be normal in the AS and MR systems. However, structural anomaly of stomata such as large, round shaped guard cells with damage in bordering regions of stomatal pores was pronounced in the DF system along with a relatively higher K+ ion concentration than in the AS and MR systems. Antioxidative status of regenerated shoots was comparable in the AS and MR systems, while a higher incidence of oxidative damages of lipid membrane as evidenced from malondialdehyde and ascorbate content was observed in the DF system. Higher oxidative stress in the DF system was also apparent by elevated activities of superoxide dismutase, ascorbate peroxidase, and catalase. Among the three culture systems, liquid culture with MR resulted in maximum shoot multiplication with little or no symptoms of hyperhydricity. Shoots in the DF system were more prone to hyperhydricity than those in the AS and MR systems. The use of matrix support such as membrane raft as an interface between liquid medium and propagating tissue could be an effective means for rapid and efficient mass propagation with little or no symptoms of hyperhydricity.

Rapid clonal propagation of Zygophyllum xanthoxylon (Bunge) Maxim., an endangered desert forage species

This study describes a reliable protocol for callus induction and rapid mass propagation of the ecologically important plant, Zygophyllum xanthoxylon (Bunge) Maxim. The optimum callus induction medium was Murashige and Skoog (MS) supplemented with 4.4 μM 6-benzylaminopurine (BAP) and 2.7 μM α-naphthalene–acetic acid (NAA), on which the callus induction frequencies from different seedling explants were all 100%. However, seedling-derived callus did not form regenerated shoots. In order to achieve shoot multiplication, shoots were developed from cultured plumules, at an average of 3.1 shoots per explant, and the regenerated shoot tips were further multiplied by subculture. The best shoot multiplication from shoot tips was achieved on MS supplemented with 5.4 μM NAA and 22.2 μM BAP after 40 d of culture. Seventy-three percent of regenerated shoots formed roots when cultured on MS supplemented with 8.6 μM IAA after 4 wk of culture. The plants that acclimatized successfully in sand flourished the following year, with normal morphology and growth characteristics.

EFFICIENT PROPAGATION BY BIOREACTOR SYSTEM OF KOREAN NATIVE GINGER IN SEOSAN REGION

To establish an efficient propagation method through airlift bioreactor system of Zingiber officinale Rosc. Korean native Seosanjong, the effect of different factors and bioreactors on cultured plantlets were investigated. The highest number of plantlets, fresh weight per plant was obtained from explants when cultured in MS liquid medium including 0.3 mg/L NAA and 2.0 mg/L kinetin for 40 days. A 10 L bottle type bubble bioreactor, compared with 250 ml Erlenmeyer flask, was more efficient, producing 4.7 plantlets or from 1.5 to 1.6 times more than did the Erlenmeyer flask. The results demonstrate a rapid mass propagation method for scaling up production of normal ginger using the airlift bioreactor.

Rapid plant regeneration through in vitro axillary shoot proliferation of butterfly pea (Clitoria ternatea L.)—a twinning legume

This study describes a reproducible protocol for rapid mass propagation of a multipurpose legume, Clitoria ternatea L., using cotyledonary node explants derived from axenic seedlings. Multiple shoots were induced in Murashige and Skoog (MS) medium supplemented with N6-benzyladenine (BA), zeatin riboside, or thidiazuron. N6-Benzyladenine at 1.0 mg l−1 (4.44 μM) was most effective for shoot proliferation. Multiple shoots were also induced in nodal segments of in vitro-raised shoots grown on MS medium containing 1.0 mg l−1 (4.44 μM) of BA. Rooting was best induced in shoots grown on half-strength MS medium with 0.25 mg l−1 (1.42 μM) of indole-3-butyric acid. Plants were acclimatized in vermicompost and established in soil where they flowered and formed mature seeds.

Rapid mass propagation and diffusion of new banana varieties among small-scale farmers in north western Tanzania

Since the seventies, factors such as declining soil fertility and the emergence of pests and diseases have reduced banana (Musa spp.) yields from about 10 to 4 t ha-1 in the Kagera region, North Western Tanzania where banana is the most important staple food. Thus, clean planting materials are required to solve the problem of pests and disease infection. To this effect, new banana varieties were introduced, multiplied locally and distributed to more than 0.5 million people since 1997. The target was set to have 1 million plants of superior varieties in the farmer's fields by March 2003 which accounts to about 1.5% of the total banana population in the region. A total of 21 varieties were introduced and evaluated for their palatability and field performance by the local communities. Fourteen varieties were multiplied In vitro. A total of 71,000 In-vitro plants were introduced in the region since 1997 and multiplied in 35 nurseries by field decapitation. These multiplication fields contain 84,000 stools and are spread over the entire region. To-date 340,000 suckers have been distributed by the project and 680,000 suckers from farmer to farmer, amounting to 1,020,000 suckers. The best performing varieties are FHIA-17, FHIA-23, SH3436-9 and Yangambi. Results of an impact assessment survey show that the superior varieties outyield the local varieties by an average of 40%. Key Words: Clean planting material, impact assessment, In-vitro, Musa spp. RESUME Depuis les années soixante-dix, des facteurs tells que la réduction des la fertilité de sols et l'émergence des pestes et maladies ont réduit le rendement de la banane (Musa spp.) de 10 a 4 t ha-1 dans la région de Kagera, au nord ouest de la Tanzanie où la banane est la nourriture de base. Par conséquent, du matériel sain est nécessaire pour résoudre le problème d'infection des pestes et des maladies. Pour ce faire, des variétés nouvelles étaient introduites, multipliées localement et distribuées a plus de 0.5 million des personnes depuis 1997. Il était ciblé d'atteindre 1 million de plantules de variétés supérieures dans les champs des fermiers vers Mars 2003 représentant 1,5% de la population bananière dans la région. Au total 21 variétés ont été introduites et évaluées pour leur goût et performance dans les champs par les communautés locales. Quarante variétés ont été multipliées in vitro. Au total 71,000 In-vitro plantes ont été introduites dans la région depuis 1997 et multipliées dans 35pépinière par découpage en champs. Un total de 84,000 coupures ont été réalisées par multiplications dans les champs et ont été disséminées dans toute la région. Aujourd'hui 340,000 rejetons ont été distribués par le projet et 680,000 rejetons d'un fermier a l'autre, pour un total de 1,020,000 rejetons. Les variétés les plus performantes sont le FHIA-17, FHIA-23, SH3436-9 et Yangambi. Les résultats de l'évaluation de l'impact ont montré que les variétés supérieures ont outré passées les variétés locales par une moyenne de 40%. Mots Clés: Matériel de plantation sain, evaluation de l'impact, In-vitro, Musa spp. African Crop Science Journal Vol.12(1) 2004: 7-17

고구마 정단분열조직으로부터 체세포배발생 및 식물체 재분화에 미치는 casein의 영향

An efficient protocol has been developed for rapid mass propagation of sweetpotato from shoot-tips derived embryogenic callus. Optimal embryogenic callus was induced from shoot apical meristem explants on Murashige and Skoog (MS) medium supplemented with 1mg/L 2,4-D. The addition of casein hydrolysate in the media increased the embryogenesis efficiency of sweetpotato. Somatic embryos were easily induced from the embryogenic callus on MS basal medium containing 300-500mg/L casein hydrolysate without phytohormon. Treatment of casein hydrolysate (100∼300mg/L) with 1mg/L 2,4-D also improved the secondary embryonic efficiency from somatic embryos below 2mm in length. Plant regeneration was achieved via somatic embryogenesis and direct organogenesis. Regenerated planlets with well developed shoots and roots on MS basal medium were successfully transferred to soil.

지황 부정근을 이용한 식물체 재분화 및 생물반응기 배양

This experiment was carried out to develop rapid mass propagation via shoot organogenesis system from adventitious roots of Rehmannia glutinosa. The induction of adventitious roots from leaf explants was most favorable to MS solid medium supplemented with 2mg/L IBA. However, the growth of adventitious roots was highest when they were cultured on 1/3 strength MS liquid medium supplemented with 2mg/L IBA. When the adventitious roots were grown in 10L bioreactor, 10g roots as initial inoculum was increased to 225g after 6 weeks of culture. The harvested roots were cultured onto solid medium to induce plant regeneration. The optimal adventitious shoot formation was observed on MS medium supplemented with 2mg/L BA. Rooting of individual shoots was induced after transfer to half strength MS medium without growth regulators. Plantlets after acclimatization were successfully transplanted in the field and no phenotypic variation was observed among them.

Rapid mass propagation of Tylophora indica Merrill via leaf callus culture

An efficient protocol has been developed for rapid mass propagation of Tylophora indica from leaf derived callus. Optimal callus was developed from leaf explants on Murashige and Skoog (MS) basal medium supplemented with 10 μ 2,4,5-T. Adventitious shoots were regenerated (85%) from the surface of the callus on MS medium supplemented with 5 μM Kinetin. Individual elongated shoots were rooted on half-strength MS medium containing 0.5 μM IBA. Regenerated plantlets with well developed shoots and roots were successfully transferred to soil. The study demonstrated a dedifferentiated callogenic propagation route via adventitious shoot development in T. indica, which could be useful for large scale multiplication of this endangered medicinal plant.

Rapid mass propagation of Chrysanthemum cinerariaefolium Vis. by callus culture and ability to synthesise pyrethrins.

Rapid mass propagation of Chrysanthemum cinerariaefolium from young flower heads was developed to compare the ability of callus, in vitro shoots and rooted plants, and original plants to synthesize pyrethrins. The ability to synthesise all six pyrethrin components increased with differentiation. Jasmolin II and cinerin II were the main products present in mother plant shoots, whereas pyrethrin I was the greater component present in callus and in vitro plants. Clonal propagation increased the pyrethrin I content compared to that of plant shoots and young flowers. Total pyrethrin content was the same in in vitro and plant shoots, but lower in these shoots than in young flowers. The pyrethrin I/pyrethrin II ratio, which is directly related to insecticidal activity, varied from 3.4 in in vitro shoots to 0.87 in mother plant shoots and young flowers.

885 PB 305 SOMATIC EMBRYOGENESIS FROM CALLUS OF THE THERMOTOLERANT `GERMAN RED' CARNATION

`German Red' is a thermotolerant cultivar of carnation (Dianthus caryophyllus) that blooms almost year-round in Texas. This study was initiated to evaluate the feasibility of inducing somatic embryos for use in gene transfer Studies and rapid mass propagation. Internodal explants, obtained from microshoots of plantlets cultured on MS medium containing 5 μM benzyladenine (BA) and 0.5 μM naphthaleneacetic acid (NAA), were used to initiate callus. Callus formation was induced on MS medium containing 3% sucrose, 0.1% casein hydrolysate and 2,4-D (1-5 μM) alone or in combination with BA (2 or 4 μM) or kinetin (2 or 4 μM). After about 5 weeks, the callus was transferred to either semisolid or liquid MS basal medium with or without kinetin and BA. Within 20-30 days, pro-embryogenic callus masses were observed. The embryos developed from white embryonic tissue and exhibited typical stages of embryogenesis. After 5 weeks, up to 70% of the cultures grown in the liquid medium with or without BA exhibited a profusion of embryo-like structures. Because only a small percentage of these developed into plantlets, more work is needed to enhance conversion frequency.

Rapid mass propagation of Chrysanthemum morifolium by callus derived from stem and leaf explants

A procedure for rapid multiplication of Chrysanthemum morifolium RAMAT cv. Birbal Sahni using leaf callus and stem (nodal/internodal) callus as well as node and apical shoots has been developed. Murashige and Skoog's medium (1962) supplemented with 2mg/1 2,4-D yielded good green calli from both leaf and stem segments within 2 weeks. About 1 cm × 1 cm callus regenerated 2-3 shoots after 3 weeks on MS solid medium supplemented with 0.1 mg/l IAA and 0.2 mg/l BAP. Each of the regenerated shoots when transferred to the same shooting medium without agar yielded about 150 new shoots, which in turn regenerated roots after another week in MS half strength or modified White's media (Rangaswamy, 1961). It has been estimated that about 10(14) plantlets could be produced in a year from one expiant following the proposed protocol.