Axillary Bud Proliferation Research Articles

Micropropagation studies of sweet orange (citrus sinensisOsbeck) cv. Blood Red

This study was conducted to study in vitro technique, micropropagation of sweet orange (Citrus sinensis Os beck) cv. Blood Red. The impact of plant growth regulators on axillary bud proliferation in Blood Red was studied by using MS basal medium added with BAP (1.0, 3.0, 5.0 and 7.0 mg l−1) and NAA (0.1, 0.5, 1.0 and 1.5 mg l−1) for rooting IBA and NAA (0.1, 0.5, 1.0 and 2.0 mg l−1 each) either alone or in combinations. The maximum percent success, number of shoots, length of shoot and minimum number of days taken to bud break were obtained with BAP (3.0 mg l−1) and NAA (0.5 mg l−1). For rooting, lower concentrations of NAA and IBA up to 0.5 mg l−1 were found better in case of maximum percent success, number of roots, length of roots and minimum number of days taken to root initiation as compared to higher levels.

An optimized method for in vitro propagation of African baobab (Adansonia digitata L.) using two-node segments

Adansonia digitata L. (African baobab), is an important multi-purpose tree, whose distribution is at present limited to wild or semi-domesticated individuals widespread in Africa. Its distribution is threatened by seedling clearance for other land use and potentially by overharvesting induced by growing commercial use of baobab fruit. Recently, efforts have been made to establish baobab domestication and conservation strategies, with mixed results due to the low germinability of baobab seeds, a factor that hinders the possibility of developing commercial A. digitata plantations. Here, micropropagation was tested as a method for clonal propagation of explants from in vivo-grown seedlings. In vitro shoot multiplication was achieved by enhanced axillary bud proliferation of sterilized two-node segments. Bud break was dependent on cytokinin supply, but the combination of 1.0 or 10.0 μM zeatin riboside and 10.0 μM indole-3-butyric acid (IBA) increased the formation of microshoots after 8 weeks of culture. Regenerated microshoots rooted successfully in in vitro nutrient medium containing 10.0 μM IBA and normally grew in a greenhouse after acclimatization.

The First Report of Stolbur Phytoplasma Associated with Phyllody of Calendula officinalis in Serbia.

Pot marigold (Calendula officinalis L.) is native to southern Europe. Compounds of marigold flowers exhibit anti-inflammatory, anti-tumor-promoting, and cytotoxic activities (4). In Serbia, pot marigold is cultivated as an important medicinal and ornamental plant. Typical phyllody, virescence, proliferation of axillary buds, and witches' broom symptoms were sporadically observed in 2011 in Pancevo plantation, Serbia (44°51'49″ N, 20°39'33″ E, 80 m above sea level). Until 2013, the number of uniformly distributed affected pot marigold plants reached 20% in the field. Due to the lack of seed production, profitability of the cultivation was seriously affected. Leaf samples from 10 symptomatic and 4 symptomless marigold plants were collected and total nucleic acid was extracted from midrib tissue (3). Direct PCR and nested PCR were carried out with primer pairs P1/16S-SR and R16F2n/R16R2n, respectively (3). Amplicons 1.5 and 1.2 kb in length, specific for the 16S rRNA gene, were amplified in all symptomatic plants. No PCR products were obtained when DNA isolated from symptomless plants was used. Restriction fragment length polymorphism (RFLP) patterns of the 1.2-kb fragments of 16S rDNA were determined by digestion with four endonucleases separately (TruI1, AluI, HpaII, and HhaI) and compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic pot marigold plants were identical to the Stol pattern, indicating Stolbur phytoplasma presence in affected plants. The 1.2-kb amplicon of representative Nv8 strain was sequenced and the data were submitted to GenBank (accession no. KJ174507). BLASTn analysis of the sequence was compared with sequences available in GenBank, showing 100% identity with 16S rRNA gene of strains from Paeonia tenuifolia (KF614623) and corn (JQ730750) from Serbia, and peach (KF263684) from Iran. All of these are members of the 16SrXII 'Candidatus Phytoplasma solani' group, subgroup A (Stolbur). Phytoplasmas belonging to aster yellows (16SrI) (Italy and Canada) and peanut witches' broom related phytoplasma (16SrII) group (Iran) have been identified in diseased pot marigold plants (1). To our knowledge, this is the first report of natural infection of pot marigold by Stolbur phytoplasma in Serbia.

Effect of Nutrient Media and Phytohormones on<i> In Vitro</i> Establishment of <i>Bambusa balcooa.</i> Roxb.

Bambusa balcooa.Roxb. is one of the most commercially important bamboo species. Some varieties of bamboo are being grown in the company’s green house. However this species is very rare and it was not cultured until now due to lack of demand and maintenance problems. In this work B. balcooa ex-plants have been established and propagated by the axillary shoot bud proliferation method. Earlier several works have been done on this plant and a protocol has been. This work emphasizes on effects of phytohormones at different concentrations and combinations upon the novel propagules that develop from the bamboo explants on placing them in multiplication media. The propagules placed in media containing cytokinins BAP (2 mg/L) and Kinetin (1 mg/L) exhibited best results of linear as well as radial growth. The other propagules in media with combinations of BAP, Kinetin and NAA were found to be dried out and did not depict noticeable growth. Comparatively next best growth pattern to the former was observed in control media that lacked phytohormones. This experiment was helpful in estimating the

Micropropagation of Laburnum anagyroides Medic. through axillary shoot regeneration

An in vitro propagation system based on the proliferation of axillary buds has been developed for Laburnum anagyroides. Culture initiation was influenced by explanting season, with the maximum response obtained from explants harvested in spring and autumn while the lowest response was noted in summer explants. The best shoot induction was observed on Murashige and Skoog medium (MS) supplemented with 2.22 μM 6-benzylaminopurine (BAP). The basal media, type of cytokinin, and explant type were the most important factors affecting shoot multiplication of L. anagyroides. Medium comprised of ½MS salts was found to be more efficient for axillary shoot multiplication compared to full-strength MS or Wood Plant Medium (WPM) when using identical growth regulators. Shoot tip explants were more responsive than nodal segments of microshoots for micropropagation. In vitro-derived shoots, >10 mm in length, were successfully rooted in medium containing ¼MS salts and 2.68 μM α-naphtalene acetic acid (NAA). In vitro-regenerated plantlets were adapted to ex vitro conditions and transferred to a greenhouse. This is the first report for successful in vitro propagation of L. anagyroides from bud explants of mature trees.

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 ± 0.8cm) and the highest response of shoot induction (95%) were recorded on MS medium supplemented with 2.0mg/l BAP. Rooting was best achieved on half-strength MS medium augmented with IBA (1.0mg/l). Half-strength MS medium supplemented with BAP (4mg/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.

Effect of Nutrient Media and Phytohormones on<i> In Vitro</i> Establishment of <i>Bambusa balcooa.</i> Roxb.

Bambusa balcooa.Roxb. is one of the most commercially important bamboo species. Some varieties of bamboo are being grown in the company’s green house. However this species is very rare and it was not cultured until now due to lack of demand and maintenance problems. In this work B. balcooa ex-plants have been established and propagated by the axillary shoot bud proliferation method. Earlier several works have been done on this plant and a protocol has been. This work emphasizes on effects of phytohormones at different concentrations and combinations upon the novel propagules that develop from the bamboo explants on placing them in multiplication media. The propagules placed in media containing cytokinins BAP (2 mg/L) and Kinetin (1 mg/L) exhibited best results of linear as well as radial growth. The other propagules in media with combinations of BAP, Kinetin and NAA were found to be dried out and did not depict noticeable growth. Comparatively next best growth pattern to the former was observed in control media that lacked phytohormones. This experiment was helpful in estimating the

Development of an efficient protocol for plant regeneration from nodal explants of recalcitrant bamboo (Bambusa arundinacea Retz. Willd) and assessment of genetic fidelity by DNA markers

The present study describes an efficient method for in vitro plant regeneration in B. arundinacea through axillary shoot bud proliferation. Nodal explants were excised, cultured on MS medium containing different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN) (0.5–5.0 mg l−1) alone and/or in combinations with KIN/BAP (0.5 mg l−1). The highest frequency (91.5 %) of multiple shoot bud induction with maximum number of shoots (85 shoots/explant) was noticed on MS medium + 3.0 mg l−1 BAP + 0.5 mg l−1 KIN. The regenerated multiple shoots were elongated on MS medium + 4.0 mg l−1 KIN + 2.0 mg l−1 gibberellic acid (GA3) with maximum shoot length (4.9 cm). The elongated shoots were transferred to MS medium containing indole-3 butyric acid (IBA; 0.5–5.0 mg l−1) alone and/or in combination with 0.5 mg l−1 KIN and BAP. Highest frequency of rooting (75 %) was obtained on half-strength MS medium + 2.0 mg l−1 IBA + 0.5 mg l−1 KIN. After hardening, the plantlets were shifted to the green house and subsequently established in the field conditions with 90 % survival rate. random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the regenerants. RAPD profiles generated from the regenerated plants were found to be monomorphic, similar to the control. Results confirmed that the regenerated plants were true-to-type in nature and the developed micropropagation protocol could be used for large scale plant production of B. arundinacea.

Improved In Vitro Propagation of Paulownia elongata, P. fortunei and its Interspecific Hybrid P. elongata x P. fortunei

Our paper presents an effective micropropagation protocol for Paulownia sp. (P. elongata, P. fortuneiand a P. elongata x P. fortunei hybrid). For in vitro culture initiation, axillary buds as well as seeds wereused. For the multiplication stage, modified Murashige & Skoog (MS) was tested, supplemented with0.5 mg/l BAP (6-benzilaminopurine) and mT (meta-topoline) at the concentrations of 0.5, 0.7 and 1mg/l. The media supplemented with BAP ensured intense proliferation of axillary buds but they alsocaused negative physiological effects (abundant callus growth at the plantlets’ base). In the P. elongata xP. fortunei hybrid as well as in P. elongata 1 mg/l mT ensured the highest proliferation and multiplicationrates, but the plantlets that resulted from the media supplemented with 0.5 and 0.7 mg/l mT were morevigorous and well developed. In P. fortunei 1 mg/l mT ensured average proliferation rates of 9.28 andaverage multiplication rates of 12.92. Wheat starch proved to be a cheap and very effective alternativegelling agent. Ex vitro rooting and acclimatization was very successful by using Jiffy7 pellets and floatingperlite, the latter being a technique elaborated at the Fruit Research Station Cluj, with acclimatizationpercentages of more than 80 % in the 3-5 cm long shoots and more than 60 % in the 2-2.5 cm longshoots. The protocol developed allowed eliminating the in vitro rooting stage in the commonly usedprocesses in the industry.

Axillary Bud Proliferation Approach for Plant Biodiversity Conservation and Restoration

Due to mainly human population pressure and activities, global biodiversity is getting reduced and particularly plant biodiversity is becoming at high risk of extinction. Consequently, many efforts have been deployed to develop conservation methods. Because it does not involve cell dedifferentiation of differentiated cells but rather the development and growth of new shoots from preexisting meristems, the axillary bud proliferation approach is the method offering least risk of genetic instability. Indeed, meristems are more resistant to genetic changes than disorganized tissues. The present review explored through the scientific literature the axillary bud proliferation approach and the possible somaclonal variation that could arise from it. Almost genetic stability or low level of genetic variation is often reported. On the contrary, in a few cases studied to date, DNA methylation alterations often appeared in the progenies, showing epigenetic variations in the regenerated plants from axillary bud culture. Fortunately, epigenetic changes are often temporary and plants may revert to the normal phenotype. Thus, in the absence of genetic variations and the existence of reverting epigenetic changes over time, axillary bud culture can be adopted as an alternative nonconventional way of conserving and restoring of plant biodiversity.

Development of Simple, Cost Effective Protocol for Micropropagation of Tylophora indica (Burm f.) Merill., an Important Medicinal Plant

Aims: The present studies were initiated to develop a cost effective protocol for micropropagation,as a mean for conservation of medicinal plant -Tylophora indica(Burm f.)Merill. The plant is threatened andneeds immediate conservation, therefore, the study was undertaken with following objective: •In vitromultiplication of Tylophora indicausing nodal axillary bud proliferation and through organogenesis of callus. Study Design:For all experiments ten repli cates were used per treatment and all the experiments were repeated three times. Data have been presented as Mean ± Standard deviation.

Micropropagation of Salacia reticulata - an Endangered Medicinal Plant

An efficient protocol of axillary bud proliferation and direct organogenesis has been developed for Salacia reticulata, a highly important medicinal plant. Over-exploitation for its antidiabetic and antioxidant properties concentrated in roots and stem has caused it to be endangered, thereby the need for its conservation. Propagation of S. reticulata in vitro is a promising way for its conservation. To develop the micropropagation protocol, the germplasm was screened for selection of a suitable ecotype with high content of mangiferin estimated with High Performance Liquid Chromatography technique. Nodal segments were cultured on MS supplemented with different growth regulators. The most efficient shoot multiplication was obtained with the supplementation of BA and IAA (3.5 + 0.5 mg/l). Elongation of the micro-shoots was achieved by subculture every 20 days. The elongated micro-shoots were efficiently rooted in vitro on half strength MS supplemented with IBA. Plantlets were successfully established in the soil in 6 - 8 weeks and were morphologically similar to those of the source plant. The protocols developed presently for direct shoot regeneration and root-induction could be successfully applied for development of high quality planting stocks. D. O. I. http://dx.doi.org/10.3329/ptcb.v23i2.17523 Plant Tissue Cult. & Biotech. 23(2): 221-229, 2013 (December)

Rapid in Vitro Regeneration and Clonal Multiplication of Thymus Bleicherianus Pomel, a Rare and Threatened Medicinal and Aromatic Plant in Morocco

This paper reports, for the first time, an efficient and reliable protocol for in vitro clonal propagation of T. bleicherianus, a rare aromatic and medicinal plant in Morocco. Initially, nodal explants collected from wild plant were used for in vitro culture establishment on Murashige and Skoog (MS) basal salts medium without plant growth regulators (PGR). Browning and contaminants were the major obstacles for in vitro culture establishment using explant derived from wild plants. To overcome this problem, the use of polyvinylpyrrolidone together with dark treatment decreased browning from 100 to 25%. The aseptic and living explants were then transferred into MS medium and all cultures were placed under light conditions. The cytokinin type and concentration were the most important factors affecting shoot multiplication. Axillary shoot bud proliferation was achieved using plant growth regulators, 6-benzylaminopurine, and kinetin and α-naphthalene acetic acid. A high number of shoot (6.39 ± 0.21) was obtained on MS medium supplemented with 4.44 μM of 6-benzylaminopurine. Regenerated shoots were rooted on plant growth regulators-free MS medium. Plantlets were successfully acclimatized to ex vitro conditions with a survival rate of 85%. All regenerated plants were morphologically identical to the mother plants. The in vitro culture system successfully established for T. bleicherianus offers a viable tool for mass micropropagation, multiplication and conservation of this very rare and threatened species.

English

Yams (Dioscorea) are well known for their medicinal use as well as nutritional values. Dioscorea plants are rapidly vanishing from nature due to their over-exploitation by human beings. In order to conserve Dioscorea plants, the present investigation was carried out with a view to regenerate plantlet of Dioscorea alata L. through in vitro culture using full strength Murashige and Skoog (MS) medium and indole-3-acetic acid (IAA) with and without sucrose.  Nodal vine segments of D. alata were used as explants and nodal segments were cultured on MS (Murashige and Skoog’s) medium supplemented with different concentrations of auxin (IAA) for axillary bud proliferation. Best shoot proliferation was observed in MS medium containing 1.5 mg/L kinetin + 2 mg/L IAA with highest rate of shoot multiplication (average of 9.90 shoots/explants). Micro shootlets were inoculated in half strength MS basal medium supplemented with different concentrations (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L) of IAA and best rooting was observed in medium supplemented with 2.5 mg/L IAA with highest root length (8.14 cm). Regenerated plants were transplanted in hardening medium containing Brick bats + Charcoal + Dried moss + Leaf molds + Soil in 1: 1: 1:1:1 proportion. Maximum survival percentage was observed as 85 to 87% after one month of transfer in hardening medium. This work proposes an economic technique for the conservation of D. alata.   Key words: Yams, Dioscorea alata L., in vitro culture, growth regulators.

Highly efficient in vitro proliferation and genetic stability analysis of micropropagated Ceropegiaevansii by RAPD and ISSR markers: A critically endangered plant of Western Ghats

Ceropegiaevansii McCann (family: Asclepiadaceae), a critically endangered plant of Western Ghats has acquired significant importance due to its medicinal implications, edible tubers, and ornamental flowers. This study deals with the optimization of axillary bud proliferation using nodal explants followed by genetic stability analysis of regenerants. Maximum number of shoots (11.6 ± 1.1) was observed on the Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (4.0 mg/l) and indole-3-acetic acid (0.3 mg/l) with 85% shoot multiplication frequency. In vitro-grown shoots were rooted best in 1/2 MS medium supplemented with indole-3-butyric acid (1.0 mg/l) with an average of 10.3 ± 0.9 roots per shoot and 92% rooting frequency. Plantlets were acclimatized best (90%) in a mixture of sterile soil, sand, and coco peat (1:2:1). Micropropagated plants were subjected to random amplified polymorphic DNA and inter simple sequence repeat markers analyses. Collectively, 759 bands were generated which were monomorphic and similar to the mother plant. Findings of this study are the first report on micropropagation and assessment of genetic stability of micropropagated plantlets in C. evansii which suggests that axillary shoot proliferation can safely be used as an effective tool for propagation and conservation of C. evansii.

English

  Micropropagation of Pinus taeda by axillary bud proliferation technique and anatomical study of adventitious roots were studied. Apical and basal segments were establishedin vitro in Murashige and Skoog (MS) culture medium. For multiple shoot induction, the explants were transferred to WV3 medium with or without 6-benzylaminopurine (BAP) (0.12, 0.25 or 0.50 µM). The effect of the culture medium and combinations of α-naphthalene acetic acid (NAA) with BAP on adventitious rooting were evaluated. The basal segments exhibited higher multiplication rates (3.7 shoots per explant) than apical segments during establishment of in vitro culture. The best rooting rate (40%) was achieved by induction of roots in water-agar (WA) culture medium supplemented with 2.69 µM NAA and 0.44 µM BAP, for 9 days, followed by transfer into GDm/2 medium. Adventitious roots can be derived from the cambium cells, from adventitious meristem, or from a callus developed at the base of the shoots. When formed indirectly, roots can be derived from peripheral cells of the callus. In treatment resulting in the best rooting rate, 58.3% of the roots were formed directly. The callus formation at stem basis did not prevent rooting and did not impair acclimatization. The plantlets were successfully acclimatized with 90% survival.                     Key words: Loblolly pine, apical segment, basal segment, rooting, vascular connection, callus.

In vitro propagation, histochemistry, and analysis of essential oil from conventionally propagated and in vitro-propagated plants of Varronia curassavica Jacq

Varronia curassavica is cultivated for the production of an essential oil useful in the pharmaceutical industry for its strong anti-inflammatory effect. Despite a growing demand, only a few studies have evaluated alternative sources of obtaining plantlets or ways to increase the yield of essential oil from this species. Therefore, this study aimed to optimize the in vitro multiplication rate and analyze the histochemistry and sesquiterpene production potential of conventionally propagated V. curassavica plants, in vitro shoots, and acclimatized plants derived from in vitro shoots. For axillary bud proliferation, Murashige and Skoog medium was supplemented with 6-benzyladenine and thidiazuron alone or in combination with naphthalene acetic acid. Axillary bud proliferation was obtained from culture of nodal or apical segments on medium containing half-strength Murashige and Skoog salts without growth regulators. After 35 d of culture, an average of five buds developed per explant. Elongation and rooting of shoots also occurred in this medium. After the transfer of rooted plants to ex vitro conditions, 100% of the plantlets survived. Histochemical analysis of leaf tissue showed the presence of lipids, acidic lipids, essential oil, phenols, and flavonoids. The essential oils from conventionally propagated and acclimatized plants were extracted by hydrodistillation and analyzed using gas chromatography. The essential oil from acclimatized plants had a similar profile to that from ex vitro plants, but with a higher concentration of the anti-inflammatory compound alpha-humulene.

Ascertaining clonal fidelity of micropropagated plants of Dendrocalamus hamiltonii Nees et Arn. ex Munro using molecular markers

Dendrocalamus hamiltonii is a giant, evergreen, clumping, multipurpose bamboo with strong culms which are mainly used for construction, handicrafts and fuel. The tender shoots are also used as food. Overexploitation of existing natural stocks coupled with harvesting of culms before seed formation, a long flowering cycle, irregular and poor seed production, short seed viability, seed sterility, limited availability of offsets and rhizomes and seasonal dependence are some of the major bottlenecks in conventional propagation of this species. Therefore, alternative methods like micropropagation can fill the gap in demand and supply of true-to-type planting material. Recently, our micropropagation protocol for rapid multiplication of D. hamiltonii through axillary bud proliferation using nodal explants from mature culms was standardized, and more than 3,000 plants were transferred to the field. However, somaclonal variations are known to appear in the in vitro-derived clones due to culture-induced stresses. Therefore, the present investigation was conducted to ascertain the effect of the length of in vitro culture age on clonal fidelity of regenerated plants using random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers. The genomic DNA samples (i.e. mother plant, in vitro-raised shoots from the 3rd to 30th passage, and in vitro-raised plants transferred to the field) were subjected to PCR amplification using 90 primer combinations (25 each of RAPD, ISSR and SSR, and 15 AFLP primer combinations) of which 76 (23 RAPD, 24 ISSR, 21 SSR and 8 AFLP) markers showed amplified DNA fragments. The 23 RAPD primers produced 162 distinct amplified DNA fragments from 2 (OPE-5) to 16 (OPE-16) fragments per primer, while 24 ISSR primers produced 181 distinct amplified DNA fragments with an average of 7.5 fragments per primer. The number of bands generated by SSR primers varied from 3 (RM-7 and RM-240) to 14 (RM-44), and the eight combinations of AFLP primers produced 369 distinct and scorable amplified DNA fragments with an average of 46.1 fragments per primer. Appearance of monomorphic bands with all the tested primer combinations confirmed the true-to-type nature of the in vitro clones of D. hamiltonii and hence the suitability of the developed micropropagation protocol for commercial-scale plant production.

Estimation of the Cloning Potential in Six Selected Genotypes of Purple Coneflower (Echinacea Purpurea L.)

ABSTRACTSix genotypes of purple coneflower (Echinacea purpurea L.) with good field performance were selected and the most suitable concentrations of benzyladenine (BA) in the culture medium were determined for the production of buds through adventitious bud regeneration and axillary bud proliferation separately. Large variation in the most suitable concentrations of BA for cultures was observed among the six genotypes and among different explants of leaf, petiole and root. Under the most suitable concentrations of BA, the strategy for production through axillary bud proliferation was much more efficient than the strategy based on adventitious bud regeneration, in five out of the six genotypes. On the basis of the obtained data, one of the genotypes could produce theoretically more than 400 000 000 plantlets in one year, two could produce more than 10 000 000 plantlets, and the other three could produce at least 100 000 plantlets.

English

The availability of a rapid vegetative amplification procedure of mass-selected superior trees greatly accelerates the development of a new tree species as a crop. This study outlined the protocol for in vitro propagation of marula nodal explants from marula seedlings. Surface sterilized explants were cultured on Murashige and Skoog media (MS) supplemented with 26 combinations of N6-benzyladenine (BA) and kinetin (KN). Shoots were elongated on MS media supplemented with low BA and KN or BA and Gibberellin A3 (GA3) concentrations. Elongated shoots were rooted on half strength MS media supplemented with indolebutyric acid (IBA) at differing concentrations. MS media supplemented with 4.8 μM BA and 2.4 μM KN resulted in average 2.5 shoots per responding explant. Moderate shoot elongation was achieved on MS media supplemented with 1.2 μM BA plus 1.0 μM KN. Maximum rooting was observed on half- strength MS media supplemented with 10 μM IBA. Marula plants were acclimatized and established in soil in the growth room at an average micropropagation rate of 0.56 per responding nodal explant. The developed protocol has potential for routine micropropagation of elite Sclerocarya birrea subsp. caffra.Randomly amplified polymorphic DNA (RAPD) analysis scoring 1845 markers showed intraclonal genetic stability between explant parent and micropropagated plants.   Key words: Anacardiaceae, axillary bud proliferation, marula, randomly amplified polymorphic DNA (RAPD), somaclonal variation