Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Shoot organogenesis and plantlet regeneration from hypocotyl-derived cell suspensions of a tree legume, Dalbergia sissoo Roxb

Summary A complete and efficient protocol is presented for plant regeneration from cell-suspension cultures of Dalbergia sissoo Roxb., an economically important leguminous tree. Factors influencing callus initiation, establishment of cell-suspension culture, callus formation from embredded microcolonies, and shoot organogenesis from suspension-derived callus were identified. Of the two different auxins tested, callus induction was better on a medium containing naphthalene acetic acid (NAA). The percentage of callus induction increased considerably when NAA at 2.0 mg l⁻¹ (10.8 μM) was added in conjunction with 0.5 mg l⁻¹ (2.2 μM) N⁶-benzyladenine (BA). Of the three different explants evaluated for callus induction, hypocotyl segments were most responsive. Friable hypocotyl-derived callus from the second subculture passage was used to initiate the cell-suspension culture. Optimum growth of the cell suspension was observed in MS medium supplemented with the same growth regulators as described above for callus induction, with an initial inoculum cell density of 1%. The plating efficiency of the microcolonies was greatly influenced by harvesting time and the gelling agent used for plating. Efficiency was highest (93%) with cells harvested at their exponential growth phase and plated in 1.2 g l⁻¹ Phytagel. Shoot organogenesis from callus cultures was higher on a medium supplemented with a combination of BA and NAA than on BA alone. Seventy-one per cent of cultures exhibited shoot-bud differentiation on a medium containing 3.0 mg l⁻¹ (13.3 μM) BA and 0.5 mg l⁻¹ (2.7 μM) NAA. Regenerated shoots were rooted on half-strength MS medium containing 1 mg l⁻¹ each of indole-3-acetic acid (5.7 μM), indole-3-butyric acid (4.9 μM) and indole-3-propionic acid (5.3 μM). Plantlets were acclimated and established in soil.     

Plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb., a timber-yielding leguminous tree species

One of the alternative methods adopted in recent years is to use biotechnological approaches for improving the tree species. The synthetic seeds offer several advantages, e.g., easy handling, storability, reduced size of propagules, and transportability. Germplasm can be effectively stored in the form of synthetic seeds. A protocol has been developed for plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb. Nodal segments collected from basal sprouts of mature trees were encapsulated in calcium alginate beads. Inability of nodal segments entrapped in calcium alginate beads to form root was a major problem. To avoid this problem, an appropriate root induction treatment was given to nodal segments for 10 days, prior to encapsulation to allow formation of root primordia. For synthetic seeds production and subsequent conversion into plantlet, nodal segments with root primordia were encapsulated using sodium alginate and calcium chloride as gelling matrix. The best gel complexation was achieved using 3% sodium alginate and 75 mmol/L CaCl2 2H2O. Maximum percentage response (85%) for conversion of encapsulated nodal segments into plantlets was achieved on 1/2-MS medium without plant growth regulators, after 25 days of culture. The frequency of conversion of encapsulated nodal segments into plantlets affected by the concentration of sodium alginate, and the presence or absence of 1/2-MS nutrients in calcium alginate beads. Plantlets with well developed roots and shoots were transferred to pots containing autoclaved mixture of peat moss and soil (1:1). Plants were also established in pots. The conversion of encapsulated nodal segments into plantlets also occurred when calcium alginate beads having entrapped nodal segments were directly sown in autoclaved peat moss moistened with 1/2-MS0 medium. Out of 60 encapsulated nodal segments, in each experiments, stored at 4 degrees C for 30 days, 44 plants developed under in vitro conditions, and 27 on peat moss moistened with 1/2-MS0.     

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree,Dalbergia sissoo Roxb

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.     

Propagation of Dalbergia sissoo Roxb. through in vitro shoot proliferation from cotyledonary nodes

A protocol is presented for micropropagation of an economically important timber-yielding forest tree, Dalbergia sissoo Roxb. (Sissoo). Multiple shoots were induced from cotyledonary nodes derived from 1-week-old axenic seedlings on Murashige and Skoog's medium containing either N ⁶-benzyladenine (BA), kinetin (Kn), isopentenyladenine (2iP) or thidiazuron (TDZ), with BA being the most effective growth regulator. High-frequency shoot proliferation (99%) and maximum number of shoots per explant (7.9 shoots) were recorded with BA at an optimum level of 8.9 μM. Concentrations of all cytokinins tested above the optimum level markedly reduced the frequency of shoot proliferation. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary node on shoot multiplication medium after each harvest of the newly formed shoots. Primary shoots were multiplied as nodal explants, and from each stem node 2 or 3 shoots developed. Thus, 60–70 shoots were obtained in 3 months from a single cotyledonary node. About 91% of the shoots developed roots following transfer to half-strength MS medium containing a combination of 5.7 μM indole-3-acetic acid, 4.9 μM indole-3-butyric acid and 5.3 μM indole-3-propionic acid. Eighty percent of the plantlets were successfully acclimatized and established in soil. Received: 1 October 1997 / Revision received: 31 March 1998 / Accepted: 7 April 1998     

Adventitious shoot regeneration in cotyledons from Japanese pear and related species

An efficient method for the generation of adventitious shoots from the cotyledons of Japanese pear and related species was developed. Cotyledons from seeds of the Japanese pear ??Okusankichi?? and the Asian pea pear ??Hokushimamenashi?? were used to determine the optimum concentrations of phytohormones in the medium. The rates of generation of adventitious shoots and the numbers of adventitious shoots per explant were highest when the media contained 5???M 1-naphthaleneacetic acid combined with 10 or 25???M 6-benzylaminopurine. These growth regulators were used to generate adventitious shoots from the cotyledons of 33 cultivars of Japanese, Chinese, Asian pea, and European pears. A high number of adventitious shoots per explant (1.3?C2.3) and high rates of regeneration of adventitious shoots (60?C76?%) were obtained from the cotyledons of Japanese pears ??Imamuraaki?? and ??Agenosho Shinanashi.?? Although pollination was not controlled, cotyledons from mother trees of old Japanese cultivars and Chinese pears tended to be more regenerable than those from other pear species. Since the rooting ability of the adventitious shoots was very low, micro-grafting was applied to obtain regenerated potted plants from adventitious shoots. Grafted regenerated plants were recovered at a rate of more than 60?%, regardless of cultivar. To our knowledge, this is the first report to evaluate shoot regeneration from cotyledons of major Pyrus species.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.)

In vitro propagation of trees using cell, tissue and organ culture is a fast emerging area. We report here the clonal propagation of Indian rosewood (Dalbergia latifolia Roxb.) from shoot callus cultures of 5 year old trees. Bud regeneration was obtained on MS media supplemented with BA and NAA. About 35% of the cultures showed organogenesis. Shoots measuring about 3–5 cm can be excised and rooted in White's medium supplemented with 1–2 mg/L IAA. Rooted plants were successfully established in soil.Abbreviations BA Benzyladenine - CM Coconut milk - 2,4-D 2,4 dichlorophenoxyacetic acid - IAA Indoleacetic acid - IBA Indolebutyric acid - K Kinetin - NAA Naphthaleneacetic acid - PVP-360 Polyvinyl pyrrolidone     

Intra-fruit seed abortion in a wind dispersed tree,Dalbergia sissoo Roxb: Proximate mechanisms

Dalbergia sissoo, a tropical tree with wind-dispersed pods, exhibits a highly positively skewed distribution of seeds per pod with predominantly only one of the four or five ovules maturing into seed. The abortion cannot be attributed to lack of pollen or resources. This study examines the hypothesis that the abortion is due to an intense rivalry among the developing sibs to gain dispersal advantage. Aqueous extract of the dominant embryos at the stigmatic end that generally develop to maturity significantly inhibited the uptake of labelled sucrose by the young developing (subject) embryos in an in vitro assay system. Extracts of tissues such as subordinate (peduncular embryos), unfertilized ovules and pod coat did not cause such inhibition. Aqueous diffusate of dominant embryos also inhibited the uptake of labelled sucrose by subject embryos. The chemical substance responsible for the inhibition appears to be heat-stable and non-proteinaceous. HPLC analysis indicated the presence of two retention time peaks, different from that of standard indole acetic acid, but with considerable overlap. We hypothesize that the compound could be an indole derivative. We propose that the stigmatic embryos have a head start due to earlier fertilization and produce a chemical that either directly (by metabolically killing) or indirectly (by preventing the uptake of assimilates) kills the proximally placed peduncular embryos.     

Direct shoot organogenesis from shoot tip explants of a highly medicinal valued tree Pterocarpus marsupium Roxb.

In Vitro Cellular & Developmental Biology - Plant - An in vitro regeneration system for propagation of a valuable medicinal tree, Pterocarpus marsupium, using shoot tip (ST) explants derived...     

Efficient plant regeneration from in vitro leaves and petioles via shoot organogenesis in Sapium sebiferum Roxb.

Sapium sebiferum Roxb. is a widespread and economically important multipurpose tree due to its high value in ornamental, and biodiesel production as well as medicine. A highly efficient in vitro plant regeneration system through direct shoot organogenesis was established for the first time from leaves and petioles of S. sebiferum. The results showed that plant growth regulators (PGRs), mechanical damage, explant orientation, explant source, and developmental stage had a strong influence on the in vitro morphogenesis of S. sebiferum. For shoot organogenesis from leaves, the highest adventitious shoot induction rate (96.67%) with 25.67 shoots per explant was obtained when mechanically damaged leaves (the first three leaf explants at the top, leaf #1–3) were cultured with the abaxial surface placed down on Murashige and Skoog (MS) medium containing 0.5 mg L^?1 thidiazuron (TDZ). For in vitro morphogenesis of petioles, the combination of 1-naphthylacetic acid (NAA) and 6-benzylainopurine (6-BA) played a key role in cell fate determination. All of the in vitro petioles produced adventitious shoots on MS medium containing 1.0 mg L^?1 6-BA and 0.1 mg L^?1 NAA, while they produced green calli on medium fortified with 0.5 mg L^?1 6-BA and 1.0 mg L^?1 NAA. The shoots were subcultured in medium fortified with 0.5 mg L^?1 6-BA and 0.1 mg L^?1 NAA for multiplication and elongation. The elongated shoots successfully rooted on half-strength MS (1/2 MS) medium fortified with 0.5 mg L^?1 indole-butyric acid (IBA) and 0.25 mg L^?1 indole-3-acetic acid (IAA), and the regenerated plantlets successfully acclimatized with a survival rate of 92.56% in the greenhouse. The genetic fidelity of in vitro regenerated plants was evaluated using inter simple sequence repeat molecular markers. The in vitro regenerated plants were found to be the true to their mother plant. This study will be beneficial for the large-scale propagation as well as the genetic improvement of S. sebiferum.

     

Direct shoot organogenesis and plant regeneration in safflower

Summary  Adventitious shoot buds were induced directly on the adaxial surface of the cotyledons of eight safflower cultivars after 14 d of culture initiation on Murashige and Skoog's (MS) medium supplemented with various levels of 6-benzylaminopurine (BA). Maximum shoot organogenesis of 54.4% with 10.2 shoots per responding cotyledon was obtained with 8.87 μM BA in the cv. S-144. Regenerated shoots were classified into three groups on the basis of their morphological features and were found to be correlated with the levels of BA. The highest number of normal shoots was obtained from 2.2 μM BA treatment. The regenerated shoots of group I (normal shoots) were rooted on half-strength MS medium supplemented with 5.3 μM α-naphthaleneacetic acid, 3% sucrose and 0.8% bacto-agar. Rooted plantlets were successfully transferred to soil and appeared morphologically normal. Histological studies revealed that shoot buds originated adventitiously from subepidermal cells.     

High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis

An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented with 4.65 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23 μM gibberellic acid (GA₃) and 0.46 μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic stability of these regenerant plantlets.     

Direct shoot organogenesis and plant regeneration in Fortunella crassifolia

An efficient in vitro regeneration system in kumquats (Fortunella crassifolia Swingle) was established. Explant types and orientations, concentrations and combinations of plant growth regulators were evaluated for their influences on efficiency of plant regeneration. It was found that the optimum explant and its orientation was epicotyl planted vertically with upper part upward, and a shoot regeneration frequency of 1.48 shoots per explant was obtained on Murashige and Skoog (1962; MS) medium supplemented with 22.19 μM 6-benzyladenine (BA). A rooting percentage as high as 74 % was obtained on 1/2 MS supplemented with 0.54 μM 1-naphthaleneacetic acid (NAA), 9.29 μM kinetin (KN), and 0.5 g dm⁻³ activated charcoal.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Adventitious shoot formation and plant regeneration from Pinus pinaster Sol. ex Aiton

Summary Pinus pinaster plants were regenerated from cotyledons excised from in vitro germinated seeds and axenically cultured on induction medium (GMD). 6-Benzyladenine (2.2 μM) induced the highest frequency of direct bud formation from cotyledons. An average of 13.1 ± 2.1 elongated shoots per cotyledon was obtained. Germination time influenced shoot induction, and the organogenic potential decreased with explant age. Cotyledons remained for 21 d on induction medium, and in order to promote adventitious shoot elongation, they were transferred to Gupta and Durzan’s DCR medium without growth regulators, containing 0.5% (wt/vol) activated charcoal and 3% (wt/vol) sucrose. Rooting was achieved by application of an indole-3-butyric acid, (396.6 μM) pulse (24 h at 4° C), followed by transfer to a sterile mixture of peat plus perlite (1:1 vol/vol). Ninety-eight to 100% of the regenerated plants were successfully acclimatized. All plants have survived after transfer to the field.