In vitro propagation of herbaceous peony (paeonia lactiflora pall.) by a longitudinal shoot-split method

A procedure for the clonal propagation ofPaeonia lactiflora Pall. cvs. Takinoyosooi and Sarah Bernhardt through shoot tip culture is described. Half strength Murashige and Shoog (1962) medium supplemented with 0.5 mg/l 6-benzylaminopurine plus 1 mg/l gibberellic acid promoted formation and growth of axillary buds. Continuous shoot multiplication was achieved by vertically splitting the shoot axis and subsequent division of elongated axillary shoots every 36 days. High frequency (57–100%) of rooting was obtained on paper-bridge liquid medium supplemented with 1 mg/l indole-3-butyric acid. Half of the rooted plantlets were established on porous soil. Thus, 700 and 300 plants of cv. Takinoyosooi and Sarah Bernhardt could be theoretically obtained from a single bud in one year.Abbreviations BAP 6-benzylaminopurine - GA gibberellic acid - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) basal medium     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.     

In vitro propagation of emetic nut Randia dumetorum (Lamb.)

An efficient protocol for in vitro shoot multiplication of Randia dumetorum (Emetic nut) has been developed. The seeds of R. dumetorum were germinated in vitro in MS medium in 5 weeks. Subsequent propagation using shoot tip as an explant was carried out in MS medium along with different concentrations and combinations of BAP (0.5-2.0) and NAA (0.0-2.0). Maximum shoot multiplication was obtained (12.7 shoots per shoot tip) in MS medium containing 1 mg/L BAP and 1 mg/L NAA. Micropropagated shoots were rooted in 1/2 MS medium supplemented with 1 mg/l IBA. This is the first report of in vitro plant propagation of R. dumetorum. In vitro grown plantlets showed a survival rate of 70% after 2 months of transplantation to natural environment.     

Reduction of vitrification in in vitro raised shoots of Chlorophytum borivilianum Sant. & Fernand., a rare potent medicinal herb

Reduction of vitrification in in vitro raised shoots derived from shoot bases and immature floral buds along with inflorescence axis used as explants of C. borivilianum, a rare medicinal herb is described. Shoot multiplication was obtained on MS medium with 2 mg l(-1) benzylaminopurine (BAP) + 0.1 mg l(-1) indole-3-butyric acid (IBA) and MS medium with 2 mg l(-1) kinetin (Kin) + 0.1 mg l(-1) 2,4-dichlorophenoxy acetic acid (2,4-D) from shoot bases and inflorescence axis respectively. Best multiplication rates were obtained from both the explants on MS medium with 2 mg l(-1) BAP. Vitrification of shoots in cultures appeared during the multiplication stage. Culture bottles with aerated caps reduced the vitrification to 80%. Reduction of BAP concentration from 2 mg l(-1) to zero during subsequent subcultures also minimized vitrification. Use of 0.5-2 mg l(-1) Kin produced healthy shoots when compared to BAP. In vitro raised shoots rooted on Knop salts containing iron and vitamins of MS medium, 2 mg l(-1) IBA and 0.1% activated charcoal. About 80% plantlets survived upon soil transfer. Scanning electron microscopic and image analyzer studies reveal the morphological structural differences between the leaves of normal and vitrified plantlets.     

Rapid propagation of Holostemma ada-kodien Schult., a rare medicinal plant, through axillary bud multiplication and indirect organogenesis

Efficient protocols of axillary bud multiplication and indirect organogenesis were established for Holostemma ada-kodien Schult. (Asclepiadaceae). Murashige and Skoog (MS) medium supplemented with 2.0 mg l^-1 N⁶-benzylaminopurine (BAP) and 0.5 mg l^-1 indole-3-butyric acid (IBA) induced an average of eight shoots per node and was the best for axillary bud proliferation. Subsequent cultures enhanced the number of shoots. The explant source of callus and the growth regulator inducing the callus exhibited significant influence on organogenesis. Callus developed from the basal cut end of the node explants differentiated more than 15 shoots on MS medium fortified with 1.5 mg l^-1BAP. Callus from internode explants developed fewer shoots than callus from the basal cut ends of node explants. Leaf-derived callus did not undergo organogenesis. The abscission of leaves and shoot tips of the developed shoots was prevented by the addition of AgNO₃ or CoCl₂, but with a concomitant significant reduction in the number of shoots. Half-strength solid MS or liquid medium with 0.05 mg l^-1 IBA exhibited the best in vitro rooting. Ninety percent of the rooted shoots survived in the field.     

In vitro multiplication of Peganum harmala--an important medicinal plant

The frequency of shoot regeneration and multiplication of P. harmala was influenced by the type of explant and kind and concentration of hormones. Of the various seedling explants, cotyledonary node exhibited maximum shoot regeneration frequency from axillary region on MS medium supplemented with 5 microM BAP. Addition of 0.1 microM NAA enhanced the efficacy of BAP for multiple shoot regeneration as well as improved the growth of shoots. BAP (5 microM) in combination with NAA (0.1 microM) was found to be the optimal for inducing an average of 4-5 shoots per explant in 75% of the cultures within 5 weeks. Replacement of BAP with other cytokinins at equimolar concentration of BAP i.e. 5 microM was not effective in inducing multiple shoots. Regenerated shoots were rooted on MS medium containing IBA (8 microM) with 80% efficiency. The plantlets were successfully established in soil where 80% of them developed into morphological normal plants.     

High frequency axillary bud multiplication and ex vitro rooting of Wedelia chinensis (Osbeck) Merr.--a medicinal plant

An efficient protocol was achieved for rapid propagation of Wedelia chinensis (Osbeck) Merr. through axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium supplemented with benzyladenine (BA; 8.87 microM) and indole-3-butyric acid (IBA; 2.46 microM) was optimal for axillary bud proliferation, which developed a mean of 8.3 shoots/node. Excision and culture of node segments from in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 days. Excision and culture of nodes in succession enhanced the number of shoots. Shoot multiplication did not exhibit decrease in the number of shoots even at 10th subculture. Nevertheless, the shoots exhibited a tendency towards stunted nature. But reduction of BA to 4.44 or 2.22 microM resumed normal growth of shoots. Half strength MS medium fortified with IBA (2.46 microM) induced the highest number of roots. All in vitro rooted shoots survived in field. Dipping of the basal end of shoots collected from multiplication medium in IBA (2.46 microM) solution for 7 days induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100%). Rooting ex vitro by direct transfer of shoots from multiplication medium exhibited 89.2 per cent survival. Use of commercial sugar and tap water and also the omission of in vitro rooting reduce the propagation cost 50-70 per cent. The protocol enables to harvest more than 50,000 plantlets within 150 days starting from a single node explant.     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid     

Successful propagation in vitro of apple rootstock MM106 and influence of phloroglucinol.

Successful in vitro propagation of clonal apple rootstock MM106 was achieved by culturing axillary buds on MS basal medium with BAP (1 mg/L), GA3 (0.5 mg/L) and IBA (0.1 mg/L). Use of liquid medium (LM) in initial cultures reduced phenol exudation to a greater extent and gave maximum sprouting percentage when transferred to solid MS medium. Phloroglucinol (PG) did not enhance sprouting of buds but increased the rate of multiplication when added in the medium. Maximum number of shoots were obtained when MS medium was supplemented with BAP (0.5 mg/L), GA3 (1 mg/L), IBA (0.1 mg/L) and PG (100 mg/L). For rooting, in vitro regenerated shoots were placed in IBA (30 mg/L) for 3 hr and transferred to solidified auxin free medium. Rooting was recorded in about 80% of shoots. Inclusion of PG in rooting medium was not beneficial but shoot cultures grown in its presence gave higher rooting percentage. Rooted plantlets showed about 70% survival rate in potting mixture of sand:soil:perlite (1:1:1).     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

Micropropagation of Madhuca longifolia (Koenig) MacBride var. latifolia Roxb.

Bud break and multiple shoots were induced in apical and axillary meristems derived from 10-d old seedlings of Madhuca longifolia var. latifolia on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l N⁶-benzyladenine (BA) singly or in combinatiobn with 1-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA). Excised shoots were rooted on half-strength MS with IBA (1.0 mg/l) after 18d of culture. Regenerated plantlets were acclimatized and successfully transferred to soil.Abbreviations BA N⁶ benzyladenine - KN kinetin - ADS adenine sulphate - IBA indole-3-butyric acid - IAA indole3-acetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

In vitro propagation ofBoswellia serrata Roxb

Anin vitro procedure for large scale multiplication ofBoswellia serrata Roxb. has been developed using cotyledonary node segments. In average 4 shoots per node were obtained on Murashige and Skoog’s (MS) medium containing 0.5 mg dm^?3 6-benzylaminopurine (BAP) and 0.05 mg dm^?3 napthaleneacetic acid (NAA) within 22 d. By repeated subculture technique 90–100 shoots per node could be obtained after 88 d of initial culture. Shoots could be rooted on MS medium containing 1/4 salts, 1% saccharose, and a combination of 0.5 mg dm^?3 indole-3-butyric acid (IBA) and 0.25 mg dm^?3 indole-3-acetic acid (IAA). Addition of antioxidants like polyvinylpyrrolidone (PVP-50 mg dm^?3) and ascorbic acid (100 mg dm^?3) in both multiplication and rooting media prevented browning of cultures. Approximately 80% of shoots rooted within 8–10 d. Rooted plantlets were kept for 15 d in culture bottles containing Soilrite^TM irrigated with a nutrient solution containing 1/4 MS salts and finally transferred to pots containing soil: Soilrite^TM (1∶1), mixture with 70% transplantation success.     

Micropropagation of Psiadia arguta through cotyledonary axillary bud culture

A micropropagation protocol for Psiadia arguta, an endangered endemic plant from Mauritius is described using 15-day old in vitro seedling explants without the radicle. MS basal medium supplemented with TDZ (0.5–1 mg/l) proved to be the most effective medium for the induction of cotyledonary axillary buds as compared to MS medium containing NAA (0.5 mg/l) or both NAA (0.5 mg/l) and TDZ (0.5–1 mg/l). In fact, after transfer to hormone free MS medium, microshoots were obtained only from seedling explants cultured on media containing only TDZ. Regenerated shoots elongated and rooted when cultured on MS8900 containing IBA (0–1 mg/l). Hormone-free MS8900 was the best medium for rooting and development of plantlets for acclimatization.     

An efficient in vitro regeneration of Ceropegia noorjahaniae: an endemic and critically endangered medicinal herb of the Western Ghats

An efficient protocol was developed for the rapid in vitro multiplication of an endemic and critically endangered medicinal herb, Ceropegia noorjahaniae Ans., via enhanced axillary bud proliferation from nodal explants. The effects of phytohormones [6-benzylaminopurine (BAP), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)] on in vitro regeneration were investigated. The highest number of shoots (18.3 ± 1.3), maximum shoot length (10.1 ± 0.8 cm) and the highest response of shoot induction (95 %) were recorded on MS medium supplemented with 2.0 mg/l BAP. Rooting was best achieved on half-strength MS medium augmented with IBA (1.0 mg/l). Half-strength MS medium supplemented with BAP (4 mg/l) and sucrose (5 %, w/v) produced an average of 5.6 flower buds per microshoots with highest (90 %) flower bud induction response. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing sterile sand and coco peat (1:1) and grown in a greenhouse with 85 % survival rate. The regenerated plants did not show any detectable morphological variation. The developed method can be successfully employed for large-scale multiplication and conservation of C. noorjahaniae.     

Micropropagation of Capparis decidua through In Vitro Shoot Proliferation on Nodal Explants of Mature Tree and Seedling Explants

In vitro clonal propagation of Capparis decidua was achieved using nodal explants from mature trees, and cotyledonary node, cotyledon and hypocotyl explants taken from the seedlings. Explants cultured on MS medium supplemented with BAP showed differentiation of multiple shoots and shoot buds in 4–5 weeks in the primary cultures. The medium with BAP (5 mg/l) was the best for shoot bud proliferation from the nodal as well as seedling explant. Shoot multiplication was best on cotyledonary node. In the nodal explants shoot multiplication was best on medium supplemented with 5 mg/l BAP and after second subculturing further multiplication of shoot buds was highest on the medium containing 3 mg/l BAP. Shoots were separated from mother cultures in each subculturing for rooting. Rooting was best achieved using 1 mg/l IBA in the medium. Rooted plantlets were transferred td earthen pots with garden soil and peat moss mixture.     

Micropropagation of Terminalia arjuna Roxb. from cotyledonary nodes

Cotyledonary node explants excised from 21 day old seedlings of T. arjuna produced multiple shoots when cultured on full strength MS or modified MS (1/2 strength major salts and Fe-EDTA) medium supplemented with different concentrations (0.1-1.0 mg/l) of BAP. Maximum 8.9 shoots/explant could be recorded after 30 days of inoculation on modified MS medium supplemented with BAP (0.5 mg/l). A proliferating shoot culture was established by reculturing the original cotyledonary nodes (2-3 times) on shoot multiplication medium after each harvest of the newly formed shoots. Shoots (each having 2-3 nodes/shoot) thus obtained were also used as a source of nodal explant that gave rise to 1-2 shoots when cultured on modified MS+BAP (0.5 mg/l) medium. Thus, 45-55 shoots could be obtained after 60 days of culture initiation from a single cotyledonary node. About 88% shoots rooted well after 15 hr pulse treatment with IBA (1 mg/l) in liquid MS medium followed by transfer to modified MS medium without IBA. About 80% of these plantlets were successfully acclimatized in plastic pots containing sand and soil mixture and 70% plantlets transferred in the field those survived even after 6 months of transplantation.     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

In vitro Clonal Propagation and Organogenesis in Spilanthes acmella (L) Murray: A Herbal Pesticidal Plant of North-East India

Multiple shoots were regenerated in MS medium using different concentrations of BAP and Kn and different combinations of BAP with IAA, NAA and IBA. Highest multiplication of shoots was obtained with BAP (0.75 mg l^?1) with 28.4 shoots per explant after 60 days of culture. Shoots rooted best on IBA (0.5 mg l^?1), numbering 48.8 per explant. Organogenesis was maximum in callus cultured on MS medium supplemented with BAP (2.0 mg l^?1) and IAA (1.0 mg l^?1).     

In vitro propagation ofBoswellia serrata Roxb

Anin vitro procedure for large scale multiplication ofBoswellia serrata Roxb. has been developed using cotyledonary node segments. In average 4 shoots per node were obtained on Murashige and Skoog's (MS) medium containing 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ napthaleneacetic acid (NAA) within 22 d. By repeated subculture technique 90–100 shoots per node could be obtained after 88 d of initial culture. Shoots could be rooted on MS medium containing 1/4 salts, 1% saccharose, and a combination of 0.5 mg dm⁻³ indole-3-butyric acid (IBA) and 0.25 mg dm⁻³ indole-3-acetic acid (IAA). Addition of antioxidants like polyvinylpyrrolidone (PVP-50 mg dm⁻³) and ascorbic acid (100 mg dm⁻³) in both multiplication and rooting media prevented browning of cultures. Approximately 80% of shoots rooted within 8–10 d. Rooted plantlets were kept for 15 d in culture bottles containing Soilrite^TM irrigated with a nutrient solution containing 1/4 MS salts and finally transferred to pots containing soil: Soilrite^TM (1∶1), mixture with 70% transplantation success.     

In vitro rooting of sentang shoots (Azadirachta excelsa L.) and acclimatization of the plantlets

Summary In vitro proliferated sentang shoots were cultured onto half-strength Murashige and Skoog (MS) medium containing combinations of 1-naphthyleneacetic acid (NAA) and indole-3-butyric acid (IBA). Sentang shoots were unable to root in the absence of both auxins. A combination of 0.5 mg NAA per and 1 mg IBA per 1 induced the most shoots to form roots. With the addition of 2.5 g activated charcoal per 1 into half-strength MS medium containing 0.5 mg NAA per 1 and 1 mg IBA per 1, roots were more numerous and longer. Substances like gelrite and phloroglucinol and sugar content which would commonly influence in vitro rooting were inhibitory to adventitious root formation of sentang. Maximal rooting of 100% was achieved in “Culture Pack,” made of fluorocarbon polymer film containing charcoal-free medium with 0.5 mg NAA per 1 and 1 mg IBA per 1. Rooted shoots were acclimatized for 4 wk. Overall survival was 80%. These findings suggest the use of Culture Pack as the culture vessels, with 0.5 mg NAA per 1 and 1 mg IBA per 1 in half-stength MS media to effectively induce roots in sentang shoots.