Shoot Bud Culture Research Articles

IN VITRO REGENERATION OF LITCHI (Litchi chinensis SONN.) THROUGH SHOOT BUD CULTURE

Attempts were made to develop protocol for in vitro regeneration of litchi through axillary shoot bud cultures. The problem of microbial contamination, phenolic exudation and media browning was controlled up to some extend by pretreatment and rapid subculturing. A high frequency (51 %) shoot induction and differentiation was obtained in litchi Gola variety axillary explants on MS medium containing GA3 and BAP (1 mg/l), Kin (2 mg/l). In vitro raised shoots better proliferated in medium containing 2 mg/l BAP. BAP had positive effect on multiplication and growth of shoots but higher concentration than 2 mg/l reduced growth. Maximum rooting frequency (66.67%) with healthier roots was obtained in shoots cultured on full strength MS medium supplemented with IBA (2 mg/l). Plants with well-developed roots were transferred to soil with survival frequency of 57%. A combination of BAP and GA3 (1 mg/l), KIN (2 mg/l) was effective in establishment of cultures. While BAP (2 mg/l) and KIN (3 mg/l) was good for better flourishing in vitro raised shoots. 6-benzylaminepurine had positive effect on multiplication and growth of in vitro shoots but concentration exceeding 2 mg/l decreased growth. Full strength MS medium containing 2 mg/l IBA under dark condition promoted rooting in in vitro raised shoots. The protocol established could prove advantageous to the horticulturists and the industry for developing trees true to the parental type.

Micropropagation of <i>Artocarpus lakoocha</i> Roxb (Monkey jack) through Shoot Bud Culture

The Lakoocha ( Artocarpus lakoocha Roxb . ), popularly known as “monkey jack”, is called as ‘ barhal’ in India, belongs to family Moraceae and comprises of many species of woody trees. The present study was aimed to develop a micropropagation protocol for Lakoocha towards commercial mass production of planting material which permits rapid and easy multiplication rates, outwits genotype barriers, offers opportunities for continuous availability of genuine planting material with economic viability. We report here a prototype system of micropropagated plantlets of Lakoocha through shoot bud culture within 6 months catering to those parameters of commercial mass multiplication. Nodal explant with an axillary bud used as explant; showed in vitro bud induction within 7.33days in MS+BAP 13.33+IAA 2.28+GA 3 0.57 µM. The maximum shoot multiplication (6.66 shoots/explant) was observed in MS medium supplemented with BAP 13.33 +IAA 1.14 µM. The microshoots were maintained in MS basal medium (BAP 0.89 µM) supplemented with Casein Hydrolysate 100 mg/l and then subsequently rooted in ½ MS medium supplemented with IBA 9.85+BAP0.89 µM+Activated charcoal 500 mg/l which resulted in 88.16% rooting. The in vitro rooted plants were primary hardened on autoclaved Sand : Soil : Peat moss (1:1:2) drenched with ½ strength MS nutrients in plastic cups covered with transparent polyethylene bags to prevent excess water loss for 3 weeks in growth room at 25±2 0 C and then transferred to shade net house (50% shade 70%±5% RH) for secondary hardening. All plants showed 88.33% survival during acclimatization. The plants that were field established showed > 85% survivability, which is under further evaluation for fruiting and quality characters.

Lodgepole pine: the first evidence of seed-based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees

Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus.

Factors influencing rapid clonal propagation of Chlorophytum arundinaceum (Liliales: Liliaceae), an endangered medicinal plant

Chlorophytum arundinaceum is an important medicinal plant and its tuberous roots are used for various health ailment treatments. It has become an endangered species in the Eastern Ghats, and a rare medicinal herb in India, due to its excessive collection from its natural habitat and its destructive harvesting techniques, coupled with poor seed germination and low vegetative multiplication ratio. In order to contribute to its production systems, an efficient protocol was developed for in vitro clonal propagation through shoot bud culture. For this, multiple shoots were induced from shoot bud explants on Murashige and Skoog's medium supplemented with 2.5-3.0 mg/L BAP, 0.01-0.1 mg/LNAA and 3% (w/v) sucrose. Inclusion of Adenine Sulphate (25mg/L) in the culture medium improved the frequency of multiple shoot production and recovered the chlorotic symptoms of the leaves. Media having pH 5.9 and 4% sucrose showed significant improvement on shoot bud multiplication and growth. In vitro flowering was observed when the subcultures were carried out for over four months in the same multiplication media. Rooting was readily achieved upon transferring the shoots on to half- strength MS medium supplemented with 0.1 mg/L IBA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the green house, successfully established, and flowered in the field. This method could effectively be applied for the conservation and clonal propagation to meet the demand of planting materials.

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

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

CYTOKININ HABITUATION FOR AUTONOMOUS SHOOT INITIATION IN PAWPAW (ASIMINA TRILOBA)

Pawpaw is native to Eastern North America and shows good potential as a tree fruit crop. Micropropagation could provide a clonal propagation method for plant production and germplasm preservation. Once established, pawpaw cultures typically produce many shoot-bud clusters that do not readily elongate. Shoot-bud cultures that had been maintained on a BA (8.9 μM) + NAA (2.3 μM) medium for over five years showed evidence of cytokinin habituation. Single shoot-buds (1.5 cm) moved to a media with or without plant growth regulators (PGR) continued to initiate new shoots at a similar rate (∼ 5 to 8 shoots per culture). Shoots on PGR medium failed to elongate shoots greater than 2 cm compared to approximately 12% for those on PGR-free medium. Single shoot-buds subcultured three times to PGR-free medium continued to initiate between 5 to 7 shoot-buds per culture and shoot elongation remained at approximately 16%. This sensitivity to cytokinin and apparent carryover effect may in part explain the recalcitrant nature of pawpaw microcuttings to rooting.

MICROPROPAGATION OF GUAVA (PSIDIUM GUAJAVA L.)

MICROPROPAGATION OF GUAVA (PSIDIUM GUAJAVA L.)

In Vitro Long-Term Storage of Date Palm

A reliable method for the long-term conservation of date palm tissue cultures is described. In vitro shoot bud and callus culture were successfully stored for 12 months at 5°C in the dark. At this conditions high percent of cultures remained viable without serious signs of senescence. However, the growth rate decreased as storage period increased. The role of sorbitol as osmotic agent in storage was examined. Health shoot bud cultures were obtained after 6 months of storage on medium containing 40 g dm−3 sorbitol. This period extended for 9 months in case of callus cultures.

Micropropagation of nodal explants from adult trees of Cleistanthus collinus.

The micropropagation of adult Cleistanthus collinus was accomplished. The nodal segments from terminal twigs of a 15-year-old tree and basal sprouts of a comparable chronological age were used for initiating shoot bud cultures. Washing explants with sterile mixtures of citric acid (520.5 µM) and PVP 40 (3.75 µM) three to four times controlled the leaching of brown inhibitory substances into the establishment medium. Axillary shoots proliferated best on MS medium containing citric acid (104.1 µM), and PVP 40 (12.5 or 25 µM) supplemented with 0.44 µM BA. The number of new shoots from nodal segments of explants placed on MS medium supplemented with 0.44 µM BA increased when the remaining lengths of nodal segments were transferred to fresh medium after the longer microshoots were harvested. The microshoots derived from basal sprouts rooted best (50%) when treated with 11.4 mM IAA for 2 min, whereas only 40% of the microshoots derived from terminal twigs produced roots after a 2-min exposure to 28.5 mM IAA. The placement of BA-soaked agar cubes on the apex-decapitated shoots controlled shoot-tip necrosis considerably. In general, explants from basal sprouts were more suitable than terminal twig explants for the micropropagation of adult trees of C. collinus.

Somatic embryogenesis from cultured epicotyls and primary leaves of soybean [Glycine max (L.) Merrill

Regeneration of several varieties of soybean [Glycine max (L.) Merrill] by somatic embryogenesis from cultured epicotyls and primary leaves has been demonstrated. Somatic embryogenesis was induced from epicotyls and primary leaves when cotyledon halves with the intact zygotic embryo axes were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg 1−1 (45.2 µM) 2,4-D. Stable, continuously proliferating globular embryo cultures (GEC) were established from small groups of somatic embryos on MS medium supplemented with 20 mg 1−1 (90.5 µM) 2,4-dichlorophenoxyacetic acid (2,4-D). Rapid multiplication of shoot tips from germinating somatic embryos was achieved on Cheng’s basal medium (CBO) containing 2.5 mg 1−1 (11.3 µM) 6-benzyladenine. Fertile plants were obtained from individual somatic embryos and in vitro propagated adventitious shoot bud cultures.

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 μM), kinetin (2.32–18.58 μM), or thidiazuron (0.1–10 μM) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 μM 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 μM). The lowest concentration of indole-3-butyric acid (4.9 μM) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.

A simple method for the recovery of multiple fertile plants from individual somatic embryos of soybean [Glycine max (L.) merrill

We describe a method for obtaining and proliferating multiple, fertile plants from somatic embryos of several experimental and commercial soybean varieties. Shoot-bud cultures were initiated by placing cotyledonary and torpedo-stage somatic embryos derived from immature seedling cotyledons onto Cheng’s basal medium (CBO) containing 0.5 to 2.5 mg/liter 6-benzyladenine (6-BA). Prolific masses of adventitious shoots were produced within 6 to 18 wk. These cultures can be propagated indefinitely with regular subcultures to CBO containing 0.5 mg/liter 6-BA. Individual shoots were separated from the clusters and were rooted on CBO medium without exogenous growth regulators. By this method any number of plants can be produced from individual somatic embryos. The risk of losing valuable genotypes (e.g., derived from in vitro selection or transformation) due to inefficient embryo germination and embryo-to-plant conversion is thus greatly reduced. Plants were established in the greenhouse and progenies were field tested. Progenies from shoot-bud culture-derived plants showed no somaclonal variation for the seven recessive marker traits or quantitative agronomic characters evaluated under field conditions.

Propagation of Valeriana wallichii DC. using encapsulated apical and axial shoot buds

Apical and axial shoot buds of Valeriana uallichii DC., an endangered medicinal plant, were successfully encapsulated in calcium alginate beads and plantlet development was observed under both in vitro (98%) and in vivo 64%; in pots with vermiculite under glasshouse) conditions. The advantages of the encapsulation procedure for clonal propagation over the presently employed shoot bud culture method are discussed in relation to economy of space, time and overall cost.

Plantlet differentiation in leaf and root cultures of birch ( Betula Pendula roth.)

The newly-formed leaves on plantlets differentiated from shoot bud cultures of Betula pendula, when excised and grown on a fresh medium produced callus from the margins or regenerated leafy shoots, roots and plantlets. After 4 weeks, upon transfer to murashige and Skoog (MS) medium supplemented with 3-indoleacetic acid (IAA) + 6-(4-hydroxy-3-methyl- trans-2-enyl)aminopurine (zeatin) + 6-aminopurine (adenine), 15–20 plantlets were produced from each explant. Likewise, the roots also showed meristematic activity at several sites, and produced nodulated callus on MS + α-naphthaleneacetic acid (NAA) + 6-(3-methyl-2-butenyl-amino)purine (2-iP) + adenine, and ultimately differentiated plantlets. Anatomical studies showed that initiation of callus takes place by meristematic activity in epidermal cells of leaves, and cortical cells of roots. Cytological investigations revealed no change in chromosomal complement.