Bamboo

Protocol was standardized for somatic embryogenesis from mature explants of Dendrocalamus hamiltonii, tracing morphogenetic events using histological techniques, biochemical analysis of amylase secretion during somatic embryogenesis. Besides, protocols were also standardized for B. multiplex and B. ventricosa using mature explants.

As the bamboos are cross pollinated and flowering cycles are long drawn, a lot of heterogeneity is observed in the seedling populations which may be important for bio-diversity conservation point of view but highly unsuitable for economic plantations. Therefore, prior selection of the seedlings in the field grown plants of D. hamiltonii was ensured in the Institute before attempting mass propagation both through nodal ex-plants in vivo as well as through tissue culture. The selections were based on the growth performance of the seedlings in the field conditions and the propagation from mature plants of known physiological age ensured better performance in the field. As rooting of micro-shoots was inconsistent, an alternative method i.e. induction of somatic embryogenesis was employed. This is first report of prior selection and use of ex-plants from mature culms of such plants for somatic embryo induction. Callus cultures were raised in BAP, NAA and 2,4-D enriched MS medium. Both friable (Fig.) and fast growing as well as nodular ( Fig.) and compact slow growing callus lumps were formed in the same media combinations comprising of MS with BAP and 2,4-D. Shoot buds were very clearly defined, but loosely held somatic embryos differentiated in the nodular callus as ivory white structures. Their germination was ensured in MS medium supplemented with high (8%) sucrose and up to 78% conversion of somatic embryos was achieved.


Fig. Embryo germination on 8 % germination Sucrose	Fig. Effect of sucrose on embryoid

Fig. Nodular callus	Fig. Friable callus

Various morphogenetic events leading to somatic embryo formation were studied in detail through histological studies. In order to find out the cells involved in callus induction, sections were cut both from the control ex-plants showing no proliferation and the ones with cells proliferating at cut ends. An abnormal divisional activity of the cells was noticed adjacent to the vascular bundles (Fig. ) whereas no such activity was found in control (Fig.). These could be the cells from phloem parenchyma located between the vessel elements, thereby, showing vascular origin. Such meristematic cells divided and redivided to form long strands of fast growing cells. Even deep seated embryogenic masses were observed in the callus tissue which eventually developed into full-fledged embryoids. Subsequently, these come to lie on the surface of nodular callus lumps and even showed signs of secondary embryogenesis from the surface cells.

Three different pathways of embryoid conversion into plantlets were clearly discerned. These were (a) individual embryoid producing rooted plantlets,

(b) embryoids lying in close proximity germinating synchronously to produce a mass of rooted plants, and

(c) germinating embryoids producing a closely appressed thalamus like structure bearing many shoots with a single thick root.

The rooted plant-lets by either of the methods described above could be transplanted directly in a potting mix, soil: sand: F Y M :: 1:1:1, under greenhouse conditions to a success rate of 82%, After 6 months in the greenhouse, plants were transferred to poly bags. Superiority of the TC raised plants was in a span of 6 years when compared to the nodal cutting raised plants of the same age.

A similar procedure of multiple shoot formation and subsequent rooting of nodal ex-plants of B. multiplex and B. ventricosa was successfully demonstrated in liquid media. It was further found that in all the species, the amount of liquid in the culture vessel was very important as excess liquid medium often resulted in the vitrification of the shoots. For rooting, bunches of shoots faired better for root induction rather than individual shoots.


Fig. Culture initiation	Fig. Multiple shoots With roots	Fig. Rhizome induction

In case of B. multiplex, since the number of sprouted buds on each node was much higher, in vitro rooting and then excision of rooted plant-lets thus produced on the nodal ex-plants, ensured faster multiplication and clonal fidelity.

Studies on starch deposition and amylase accumulation during somatic embryogenesis in Dendrocalamus hamiltonii

In spite of the fact that the aleurone cells of starchy cereal seeds play the most significant role in α-amylase production, a controversy still exists between scutellum versus aleurone, the concept which has remained unresolved. In monocots, zygotic embryo is protected and nourished by an endosperm.

In the present study, starch deposition and amylase accumulation was noticed during somatic embryogenesis in stem callus of D. hamiltonii. SEM studies revealed that starch grains were clearly visible in the scutellum during the maturation stage of the somatic embryo. As the somatic embryo developed further, the scutellum reduced with corresponding increase in amylase. The role of scutellum in somatic embryos for starch deposition and amylase accumulation was established.

Amylase activity was estimated at various developmental stages of somatic embryos (Fig. 6). The amylase activity was absent in the callus induction, callus proliferation and globular stages of the somatic embryo development. However, it increased with elongation of somatic embryos with concomitant development of the scutellum (3.62 units) and also during early germination of somatic embryos (4.06 units). However, there was an abrupt decline in amylase activity at the embling stage (Fig.6).

Fig. α-Amylase activity during different development stages of somatic embryo development

The role of scutellum in zygotic embryo is well known but its role in somatic embryo had remained unresolved. It is established that α–amylase accumulation and starch deposition during somatic embryo development are taken over by the scutellar tissue in the absence of aleurone layer. Histological and SEM studies revealed starch deposition in scutellar tissue in mature somatic embryos. As the embryos germinated, the size of scutellum reduced with α–amylase activity.

During development of the somatic embryos, genes for starch synthesis and depositions were probably triggered. As there was no specific organ (like endospermic tissue in zygotic embryo) available for starch deposition, scutellum probably took over the function of starch deposition and also simultaneously synthesized amylases which were much needed for solubilization of stored starch into sugars. Thus, vascular strands could be the channels for transporting solubilized food reserves from the scutellum to the somatic embryo proper as evidenced anatomically (Fig. 3). Therefore, it could be concluded that in the absence of aleurone tissue in the somatic embryo of D. hamiltonii, scutellum plays an important role in amylase accumulation and act as a site for starch deposition.

Under this network programme, Dendrocalamus hamiltonii, D asper and Bambusa bambos species were allotted to IHBT for field collection, maintenance (in the nurseries/polyhouses) and raising the plants through nodal cuttings as well as tissue culture.

Around 11000 seedlings were collected during April- September, 2005 from different locations in Kangra and Hamirpur where this species is predominantly growing. These seedlings were maintained in the polysleeves under polyhouse.

About 30,000 nodal cuttings covering an area of 3.5 ha within the Institute’s command area. The sprouted seedlings will be supplied to various centers in the country for cultivation.

Refinement of existing protocols was done subsequently for efficient and largescale multiplication and ex vitro establishment of tissue culture raised plants of D. hamiltonii, D. asper, Bambusa bambos. Seeds were germinated in poly-sleeves and seedlings transplanted in sleeves, kept under poly tunnels

Five stock plants were procured from FRI, Dehradun and fresh cultures using nodal explants have been raised. Although flowering was noticed in one of the stock plants but no seed set was observed so far. Around 850 plants are ready for field transfer.

Fresh seeds of B. Bambos were procured from the Kerala Forest Research Institute, Peechi, a private nursery from Dehradun and IFCTB, Coimbatore and kept in plastic pots for germination but no germination was observed so far. But seeds procured from Department of Forestry, Aurangabad, Maharashtra state (obtained one year before) have germinated and produced 600 field transferable seedlings. Meanwhile, more than 100 plants have also been raised using nodal cuttings in the polysleeves. Aseptic cultures from nodal segments of field grown plants have been established and bunches of shoots are transferred to the rooting media.

In Bambusa nutans aseptic cultures were established in MS medium containing 3% sucrose using nodal explants of precocious branches of field grown plants. Multiple shoot formation was achieved on MS medium containing varied concentrations of PGRs. Furthering the work, shoots formed in TD2 were later transferred to BAP (1mg/l) after a period of one month and simultaneously on the medium without cytokinins. Prolific shoot formation was achieved. Different concentrations of auxins IBA, either alone and or in combination with cytokinins were used for rooting experiments. Segments from sprouted buds were used as explants for initiating embryogenic callus. This appeared as compact and slow growing. Earlier experiments with Dendrocalamus giganteous showed great difficulty in establishment of aseptic cultures. An alternative source for explants was young spikelets. Different combination and concentrations of cytokinins and auxins were employed utilizing such floral buds which got converted into vegetative shoots within three weeks. Such shoots were being multiplied further.

Propagation of Bambusa nutans A. Multiple shoots formation B. Induction of somatic embryos C. Germinating embryoids to form plantlets and Dendrocalamus giganteous D. Aseptic culture initiation from young spikelets E. Vegetative shoot formation from spikelets F. Beginning of the callus formation for somatic embryo induction.

RAPD analysis revealed genetic diversity assessment with 42 RAPD primers. Furthering the work was done by adding more RAPD markers for larger coverage of genome. Genetic diversity assessment with other marker systems such as AFLP and STMS is being done. So far, Amplified fragment length polymorphism (AFLP) was standardized and screening of the different primer pairs to find suitable primers for the molecular characterization is in progress.

DNA fingerprinting in this project was aimed at the development of molecular markers for identification and assessment of genetic diversity of Ochlandra travancorica and Leucaena leucocephala. Genomic DNA was isolated from 32 genotypes of O. travancorica following the standardized protocol.
From 175 RAPD primers screened, 45 primers produced reproducible RAPD profiles. Cluster analysis of data generated with 40 reproducible primers revealed diversity among the genotypes. A representative RAPD profiles of primers OPV 16 and OPV -18 and dendrgram generated with the 40 primers is depicted in figures

Fingerprints of 24 plants of Ochlandra travancorica of developed by RAPD primers OPV 16 (A) and OPV 18 (B) M = 100 bp ladder plus standard

Fig 4.10: Dendrogram showing genetic similarity of 32 individuals of Ochlandra generated with the 40 RAPD primers. Scale indicates the Jaccard’s similarity coefficient.

Seeds of Ochalandra travancorica were procured from Kerala Forest Research Institute (KFRI), Peechi and germinated in plastic pots. Nodal explants were used to raise aseptic cultures. Similarly, seeds of Leucacena leucocephala were collected from Jammu, Punjab, Haryana and Himachal Pradesh for germination under polyhouse and explants from selected seedlings were multiplied in vitro.

Genetic transformation of somatic embryos and embryogenic calli was attempted using both Agrobacterium and biolistic methods. For both methods, levels of selection antibiotics, kanamycin and hygromycin were screened. For biolistic, yellowish green clumps of embryogenic calli were bombarded with plasmid DNA harbouring osmotin and nptll genes using different parameters. The bombarded embryos were first cultured on regeneration medium i.e. MS supplemented with 2,4-D, BA and sucrose (3 %) and transferred to embryo germination medium. The putative transformants (emblings) were placed on selection medium containing 50µg/ml kanamycin and only one transformant that could be screened was multiplied. PCR analysis of the transformant yielded the requisite amplification product of 730bp fragment of osmotin gene. Two plantlets that were transferred to the greenhouse were showing vigorous growth. For Agrobacterium tumefaciens mediated transformation, the strain EHA 105 harbouring gus and hpt genes was used. Preliminary experiments showed that bactericidal antibiotic (i.e. cefotaxime) at 250 µg/ml concentration was lethal to growth of Agrobacterium. Medium supplemented with cefotaxime supported better embryoid growth as compared to other antibiotics.

		The promising ones were selected on the basis of phenotypic characters viz., number of culms produced/year, length and diameter of the culms at 3rd internode from base. Nodal cuttings from secondary and tertiary branches were collected from plantation raised at IHBT. Single node cuttings were planted (15-20 cm) deep in horizontal furrows and covered with soil during March-April 2004. The nodal buds sprouted within 2-3 weeks to produce plants. New culms emerged after monsoon and were ready for uprooting and transplantation. Similarly, for micro-propagation, single node explants from field selected mature elite seedlings viz., HP-1986-1, and HP-1986-2 were used for both clonal propagation by axillary proliferation and raising embryogenic slow growing nodular callus. Embryoids developed on these calli germinated into rooted plants, hardened in poly/green houses in a suitable potting mix.The performance of TCPs vis-à-vis the cutting raised plants was compared in the fields and TCPs were found superior during six years of growth. The same procedure was followed for other bamboo species under study.
Seedlings and nodal cuttings of D. hamiltonii

Nodal bud proliferation D. hamilltonii