Amita Bhattacharya

The roles of plant phenolics in defence and communication during Agrobacterium and Rhizobium infection.

Phenolics are aromatic benzene ring compounds with one or more hydroxyl groups produced by plants mainly for protection against stress. The functions of phenolic compounds in plant physiology and interactions with biotic and abiotic environments are difficult to overestimate. Phenolics play important roles in plant development, particularly in lignin and pigment biosynthesis. They also provide structural integrity and scaffolding support to plants. Importantly, phenolic phytoalexins, secreted by wounded or otherwise perturbed plants, repel or kill many microorganisms, and some pathogens can counteract or nullify these defences or even subvert them to their own advantage. In this review, we discuss the roles of phenolics in the interactions of plants with Agrobacterium and Rhizobium.

Recent Advances of Tea (Camellia Sinensis) Biotechnology

Tea is one of the most important non-alcoholic beverage drinks worldwide and gaining further popularity as an important ‘health drink’. It is served as morning drink for 2/3rd of world population daily. Although conventional breeding and propagation contributed significantly for last several decades for varietal improvement, due to the limitations of conventional breeding coupled with the demand for increasing productivity with lower cost of production, application of biotechnology becomes an alternative approach. Therefore, apart from a dozen of tea research institutes globally, several other groups are working on tea and related genera that have registered many valuable information with several achievements. The present review deals with progress in-depth of various aspects of biotechnological works such as micropropagation and alternative approaches, cell and organ culture techniques, genetic transformation, DNA markers as well as organelle genome and gene cloned from tea and related genera which will be valuable information for the workers working on various aspects of Camellia biotechnology.

An efficient liquid culture system for tea shoot proliferation

The efficiency of thidiazuron in promoting tea shoot proliferation in liquid medium was evaluated. As compared to 6-benzyl adenine which induced hyperhydricity in the proliferated shoots in liquid medium, a progressive increase in the multiplication rate together with healthy shoot growth was achieved when thidiazuron (2.5 to 5.0 μM) was used instead of 6-benzyl adenine. Of the different liquid volumes compared in 250 ml Erlenmyer flasks, 20 ml was the most effective. While an increase in the multiplication rate coupled with normal but healthy shoots was observed under static and agitated conditions at this volume of liquid medium, hyperhydricity was induced in 50 ml liquid medium. Therefore, 20 ml static liquid medium with subculture periods at an interval six to eight weeks seems to be a cost and labour effective process as compared to the existing protocols involving solid media with subculture periods at 4 weeks interval.

Problems and possibilities of monocot transformation

Biotechnological improvement of monocots is often hampered by the lack of efficient regeneration systems, requisite wound responses and low cell competence. Despite these limitations, the biolistic and Agrobacterium methods have been successfully used to produce several transgenic monocots by adjusting the parameters that govern efficient delivery and integration of transgene(s) into plant genome. It is now possible to transform even difficult monocots using tailor-made gene constructs and promoters, suitable A. tumefaciens strains and a proper understanding of the entire process. This success has been reviewed in the present article and a special emphasis was laid on the measures that were taken in overcoming the difficulties that arise due to the differential responses of monocots and dicots. This information is necessary for biotechnological improvement of still newer monocotyledonous plants that have been hitherto difficult to transform.

Factors affecting germination and conversion frequency of somatic embryos of tea [Camellia sinensis (L.) O. Kuntze]

Summary The cause of poor and abnormal germination of tea somatic embryos was investigated with respect to (a) the different factors that affect reserve mobilisation viz. chilling, desiccation or GA3 and (b) those that affect the maturation process and reserve accumulation viz. ABA. Tea somatic embryos were sensitive to desiccation and their normal development or germination could not be evoked by external agents like chilling and GA3. Supplementation with external sources of nutrient precursors and readily available forms of carbohydrates like sucrose or maltose together with trans-cinnamic acid improved the germination of the somatic embryos significantly.

Agrobacterium-mediated genetic transformation of tea leaf explants: effects of counteracting bactericidity of leaf polyphenols without loss of bacterial virulence

Tea is one of the major crops in Asia and Africa, and its improvement by genetic modification is important for economy of many tea-producing regions. Although somatic embryos derived from cotyledon explants have been transformed with Agrobacterium, the leaves of several commercially important tea cultivars have remained recalcitrant to transformation, largely due to bactericidal effect of polyphenols that are exuded by tea leaves in vitro. Moreover, the commonly used polyphenol adsorbents and antioxidants cannot overcome this problem. Leaf explants, however, are more desirable than cotyledon-derived somatic embryos, especially when it is necessary to further improve a selected elite and also retain its superior traits. Thus, we developed a procedure for Agrobacterium-mediated genetic transformation of tea leaf explants which is based on the presence of l-glutamine in the co-cultivation medium. We then showed that the transformation process is facilitated via a protective action of l-glutamine against bactericidal effects of leaf polyphenols without affecting the bacterial virulence (vir) gene expression.

INDUCTION OF SYNCHRONOUS SECONDARY SOMATIC EMBRYOGENESIS IN CAMELLIA SINENSIS (L.) O. KUNTZE

Summary Nutritional effect of nitrate salts of potassium and ammonium, together with different concentrations of sulphate salts of aluminium, potassium, magnesium, and ammonium on secondary somatic embryogenesis, wereinvestigated. Nitrate salts of potassium (9.39 mmol/L) and ammonium (10.31 mmol/L) with only 1.5 mmol/L potassium sulphate produced maximum number of synchronous secondary embryos (i.e. 20-25 secondary embryos per primary embryo in 91.6 percnt; responsive explants). Of the different factorial combinations of glutamine, BAP, and IBA tested, maximum number of synchronous secondary embryos developed on a medium supplemented with 8.88 μmol/L BAP, 0.98 μmol/L IBA and 10 mmol/L glutamine. Synchronous and normal development of secondary embryos could thus be obtained when optimal concentrations of PGRs, glutamine, nitrates, and salts of potassium sulphate were combined together. Germination of the embryos (up to 52 percnt;) was acheived only when sulphate salts of potassium were removed from the medium.

Propagation of Picrorhiza kurroa Royle ex Benth: An important medicinal plant of Western Himalaya

This study is aimed at developing propagation methods and ex situ conservation for Picrorhiza kurroa, an endangered medicinal plant of western Himalaya. Regeneration using leaves from mature plant of characterized germplasm is beneficial because the source plant is not damaged. A regeneration protocol was standardized by using leaves from aseptic shoot cultures, raised from ex vitro leaves. Maximum regeneration percent (94.33) and significantly higher shoot number (38.0) was evident in middle portion of leaf at 2.32 µM of kinetin (Kn). Abaxial surface that was in touch with the medium was more responsive as compared to adaxial surface. The time of exposure to thidiazuron (TDZ) was emphasized as 15 days interval, gave the best response in terms of shoot number (42.0). For shoot multiplication, Kn at 2.32 µM was optimum. Microshoots with well developed root system were obtained in MS basal medium after 4 weeks. Incubation of cultures at low temperature (15°C) for ten days enhanced the survival percent under green house conditions and could be correlated with the development of thick cuticle and well differentiated leaf tissues (palisade and spongy parenchyma). Flow cytometric analysis was performed to check the genetic stability of in vitro plantlets. In a parallel study, seed progenies of these germplasm were raised under ex situ conditions. Its reproductive cycle was also studied for successful domestication.

Seed development in Camellia sinensis (L.) O. Kuntze

Seed development of tea was studied to identify the maturity index and the optimal time of seed collection. After harvest, the moisture content (28-30% fresh weight basis) of mature seeds, which germinated 100%, declined progressively (19% moisture content) after shedding, with a decrease in seed germination and viability. However, this viability loss could be prevented to some extent by storing seeds within intact fruits. The maximum rate of seed dry matter accumulation coincided with the accumulation of starch in the embryos and seeds at stage 8, the embryo maturation phase. Although the embryo abscisic acid (ABA) content was highest at stage 8, free ABA declined in the tea embryos throughout the remainder of the seed maturation cycle.

An efficient protocol for somatic embryogenesis and its use in developing transgenic tea (Camellia sinensis (L.) O. Kuntze) for field transfer

With the increasing popularity of tea (Camellia sinensis (L.) O. Kuntze) as an important beverage drink throughout the world, the demand for high quality and high yielding varieties is also rising. Therefore, genetic improvement of tea through biotechnological means has become a necessity. Improvement via Agrobacterium tumefaciens mediated transformation holds potential. However, there is an urgent requirement for an efficient in vitro system of regeneration for its successful implementation.