Trees are an integral part of human life, and a vital component of biodiversity. Forest trees in particular are renewable sources of food, fodder, fuel wood, timber and other valuable non-timber products. Due to the rapid growth of population and the human desire to progress, there has been a tremendous reduction in forest cover from the earth’s surface. To maintain and sustain forest vegetation, conventional approaches have been exploited in the past for propagation and improvement. However, such efforts are confronted with several inherent bottlenecks. Biotechnological interventions for in vitro regeneration, mass micropropagation and gene transfer methods in forest tree species have been practised with success, especially in the last decade. Against the background of the limitations of long juvenile phases and life span, development of plant regeneration protocols and genetic engineering of tree species are gaining importance. Genetic engineering assumes additional significance, because of the possibility of introducing a desired gene in a single step for precision breeding of forest trees. There are no comprehensive and detailed reviews available combining research developments with major emphases on tissue culture and basic genetic transformation in tree species. The present communication attempts to overview the progress in tissue culture, genetic transformation and biotechnological applications in the last decade and future implications.

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Progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview

IT is rare, even in these days of specialized studies, that circumstances permit an investigator to present a 'first book' on pioneer work in any subject. This opportunity has come to Dr. White, and he has taken full advantage in the presentation of an admirable book. In the past, text-books of, tissue culture have given the quite erroneous impression that only animal biologists have any interest in the subject or could derive anything of value from its study. Dr. White presents the case of the plant biologist, based on 457 references, with commendable force. He compresses into this relatively small volume the 'high spots' of plant tissue culture up to the present, in which he has himself played an outstanding part, and points out wide fields of interest for future research. A Handbook of Plant Tissue Culture By Dr. Philip R. White. Pp. xiv + 277. (Lancaster, Pa.: The Jaques Cattell Press, 1943.) 3.75 dollars.

A Handbook of Plant Tissue Culture

A rapid micropropagation system was established forHolostemma annulare (Roxb.) K. Schum., (H. ada-kodien R. Br. ex Schult; Asclepiadaceae), a rare medicinal plant. Shoot tips (0.5–0.8 cm) and terminal and basal nodes (1.0–1.5 cm) harvested from actively growing shoots of conventionally raised plants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). Multiple shoot formation (3.8) was observed in 68% of basal nodes cultured on medium with optimum concentration of 4.43 μM BA and 0.54 μM NAA after 8 wk. Terminal nodes were not suitable for inducing multiple shoots. Irrespective of the orientation (vertical/horizontal), all shoot tip explants responded with a single shoot in all the combinations of plant growth regulators tried. Effects of other cytokinins (kinetin and 2-isopentenyladenine) and auxins [indole-3-acetic acid and indole-3-butyric acid (IBA)] to enhance the regeneration potential of basal nodes were analyzed. Shoots were multiplied by subculture of basal nodes and stumps (the original explant tissue free of shoots, but with remnant axillary, meristem and two or three protruding buds) in a reduced concentration of BA (2.21 μM) and NAA (0.27 μM). Liquid medium for multiplication was found to be ineffective due to a high degree of hyperhydricity. To make the multiplication process cost effective, culture bottles with polypropylene, caps were used for multiplication. The best root induction (75%) and survival (80%) was achieved on 0.5 strength MS medium supplemented with 1.48 μM IBA. Field-established plants had uniform growth habit traits in terms of height of plants and number, length, and weight of the tuberous roots.

In vitro propagation of Holostemma annulare (Roxb.) K. Schum., a rare medicinal plant

An efficient procedure for the conservation of mature American elm (Ulmus americana L.) trees that have survived the epidemics of Dutch elm disease and are potential sources of disease resistance i...

In vitro conservation of American elm (Ulmus americana): potential role of auxin metabolism in sustained plant proliferation

An efficient in vitro process for rapid clonal propagation of a 40-year-old tree of Azadirachta indica employing nodal stem segments was developed. Season of collection and maturity of explants showed direct influence on bud-break. Nodal stem segments collected during the month of April gave best response. Maximum bud-break (78.6–81%) was obtained when middle order nodes (3rd or 4th node from apex) were taken. Amongst different cytokinins used, 6-benzylaminopurine (BAP) at the concentration of 1.11 μM was found most effective in inducing multiple shoots, whereas inorganic and organic constituents of the medium influenced growth and general condition of proliferating shoots. On an average 3.1 shoots per explant were regenerated in modified Murashige and Skoog medium supplemented with 1.11 μM BAP, 1.43 μM indole-3-acetic acid (IAA) and 81.43 μM adenine hemisulphate. Isolated shoots were rooted in presence of 2.46 μM indole-3-butryic acid (IBA). Root induction took place in 8–10 days with 100% rooting. The in vitro-raised plantlets were successfully transplanted in potted soil and finally grown under field conditions with 100% survival. The genetic fidelity of such in vitro-raised field-grown plants was ascertained by random amplified polymorphic DNA (RAPD) markers. Furthermore, the azadirachtin content of in vitro-cloned plants was found comparable to the mother tree proving their chemical stability also. The protocol developed holds good for in vitro cloning of mature elite neem trees.

Rapid in vitro cloning of a 40-year-old tree of Azadirachta indica A. Juss. (Neem) employing nodal stem segments

Cleistanthus collinus Benth. was micropropagated using nodal explants on MS medium supplemented with 2.2 μM benzyladenine (BA). April to June was the best time for initiating shoot cultures. Shoot proliferation was enhanced when the BA concentration was lowered to 1.1 μM. Rooting was achieved on half-strenth MS medium with 22.8 μM indole-3-acetic acid for 7 days and continuous darkness for the first 72 h of the 7 days.

In vitro micropropagation from nodal segments of Cleistanthus collinus

The micropropagation of neem (Azadirachta indica) was accomplished by culture of buds from crown branches of a mature tree, basal-sprouts of another mature tree and a single juvenile plant. Cultures derived from these three different sources showed significant variation in in vitro response. In case of the crown and basal-sprout explants, addition of 12.5 µM PVP-40 in the establishment medium controlled leaching of phenol growth inhibitors. Phenolic leaching was not observed in juvenile explants. DKW medium (with 0.22 µM BA) was significantly better than MS for shoot proliferation. Shoot cultures of crown branch origin did not elongate and eventually died after the third subculture. In the presence of 4.9 µM IBA in half-strength DKW, 90% of the shoots and 100% of basal-sprout and seedling explant origin, formed roots. Plantlets from both explant types showed 90% survival after acclimatization.

In vitro Clonal Propagation of Neem (Azadirachta indica)

In vitro regeneration of Curcuma angustifolia Roxb. was achieved through shoot meristem culture. The shoot buds (2-3 cm long) from rhizome were inoculated on MS medium supplemented with 3.0 mg/L BAP for initiation and elongation of shoots. As a result, 1.87±0.28 shoots per explant were produced These shoots were transferred on MS medium supplemented with 3.0 mg/L BAP and 25 mg/L adenine sulfate for further shoot multiplication. About 6.9±0.69 micro-shoots per explant were produced with in 6 wk. The roots appeared from shoots on shoot establishment as well as multiplication media. The rooted plants were transferred to pots and acclimatized, which showed 83% survival with normal growth.

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In vitro propagation of tikhur ( Curcuma angustifolia Roxb.): A starch yielding plant

Axillary bud cultures were initiated from 3 types of nodal explants of Lagerstroemia parviflora. The cultures derived from explants of seedlings, terminal twigs of a 50-year-old tree and basal-sprouts of another 50-year-old tree showed significant variation in responses at establishment, shoot proliferation and rooting stages. All the 3 types of explants exuded phenolic substances from their cut ends. The exudation was checked by suspending them in a solution of 25 µM PVP 40 and 522.5 µM citric acid; and by the addition of 100 µM PVP 40 and 522.5 µM citric acid in establishment medium. Leaching continued upto rooting stage, therefore, PVP 40 and citric acid were added in MS medium used for successive transfer and rooting of microshoots. Seedling and basal-sprout explants placed on MS medium with 0.44 µM BA showed maximum shoot lengths 1.45 cm ± 0.13 and 1.16 cm ± 0.22, respectively. Tree explants exhibited best axillary shoot elongation (0.8 cm ± 0.07 cm) on MS medium without plant growth regulators. The cultures derived from seedling and basal-sprout explants could be successfully maintained upto 6th successive transfers, whereas, those derived from tree explants died after 3rd transfer. Microshoots obtained from seedling and basal-sprout explants showed 10% rooting on MS medium supplemented with 4.9 µM IBA.

Micropropagation of Lagerstroemia parviflora through axillary bud culture

We report a protocol for somatic embryogenesis and plantlet regeneration of Buchanania lanzan Spreng (Family—Anacardiaceae), which is a tropical fruit tree widely distributed in the dry forests of India. Calluses were initiated from immature zygotic embryos cultured on Murashige and Skoog (MS) medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyladenine (BA) and/or 1-naphthaleneacetic acid (NAA). The highest frequency (60%) of somatic embryo induction was obtained in cultures grown on MS medium fortified with 4.53 μM 2,4-D, 5.32 μM NAA and 4.48 μM BA. The medium supplemented with 15 μM abscisic acid (ABA) was most effective for maturation and germination of somatic embryos. This is the first report on somatic embryogenesis in B. lanzan, which may be helpful for in vitro propagation, ex situ conservation and genetic manipulation of this species.

Vijaya Koche

Papers

In vitro micropropagation from nodal segments of Cleistanthus collinus

In vitro Clonal Propagation of Neem (Azadirachta indica)

In vitro propagation of tikhur ( Curcuma angustifolia Roxb.): A starch yielding plant

Micropropagation of Lagerstroemia parviflora through axillary bud culture

Somatic embryogenesis in buchanania lanzan spreng