Plant Tissue Culture. Dr. Alain Lemansour UAE University Date Palm Development Research Unit Dept.

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1 Plant Tissue Culture By Dr. Alain Lemansour UAE University Date Palm Development Research Unit Dept.

2 What is it? Tissue culture is the term used for the process of growing cells artificially in the laboratory Tissue culture produces clones, in which all product cells have the same genotype Explant Source: Shoot tips, stem nodes, Leaf discs, flower buds

3 What is needed? Appropriate tissue (some tissues culture better than others) A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture

4 What is Needed Growth regulators Auxines and cytokinins Amino acids and vitamins Frequent sub culturing to ensure adequate nutrition and to avoid the build up of waste metabolites

5 Nutrient Media for Plant Tissue Cultures Provide water Provide mineral nutritional needs Provide vitamins Provide growth regulators Access to atmosphere for gas exchange Removal of plant metabolite waste

6 Major Components Salt Mixtures Organic Substances Natural Complexes Inert Supportive Materials Growth Regulators

7 Propagation of Date Palm by Tissue Culture

8 The date palm (Phoenix dactylifera) is cultivated in over 40 countries with approximately hectares,annually producing some seven million metric tons of fruit (FAO, 2010) with approximately 144 Million of Date Palm Trees. The date palm is widespread in North Africa, the Near and Middle East and Southern Asia. During the last century, production has been introduced into some new world locations including the USA, South Africa, China, India and Australia

9 In general, the geographical distribution of commercial date production is limited to areas which can be described as arid or semi-arid and where there is water supply. The best date growing districts are characterized by having long, hot, dry summers with minimal summer/autumn rainfall. This hardy plant species also provides a high tolerance to salinity, drought and extremes in temperature thus ensuring better food security outcomes.

10 UAEU laboratory is one of the leading laboratory in the world with a proven expertise in micro-propagation through tissue culture after 28 years of Research & Development and thousands of plants currently in production in the Middle east, Persian and North African countries. Average growth rate of date production these last 50 years was above 6% per annum and we will see that there are many reasons which will sustain that growth rate. Additionally, date palm trees face many challenges (disease, war, natural disaster) while more food will be needed to feed humans and animals. Therefore, more than 8 Million new trees need to be plant each year which is much over the existing capacities of tissue culture production centers.

11 PROPAGATION OF DATE PALM:TRADITIONALVS IN VITRO 1. Sexual Propagation, where new plantlets originate from the sexual embryo in the seed. The resulted seedlings differ considerably in fruit quality, harvesting time and production potential. Due to the high degree of genetic heterozygosity and dioecious nature of date palm, sexual propagation method cannot be used for propagation the cultivars of interest in a true-to-type manner. Hence, the technique is very useful in breeding programs and selection from among the progeny can lead to developing some elite palms with interesting traits. However, this method cannot be used to propagate elite or select genotype since the progeny will be quite variable because of the highly heterozygous character of date palm. Moreover, half of the progeny will be composed of male trees and half female tree, Female seed-derived plants will produce variable fruits which are generally of inferior quality.

12 2. Vegetative propagation, which is carried by : Offshoot propagation (traditional method). Date palm is the only species from the Arecaceae family that produce offshoots (develop from axillary buds on the trunk of the mother plant). Offshoots are true to type to the parent plant and consequently the fruit produced will be of the same quality and uniformity. This method of propagation is limited since mother plant produces 15 to 20 offshoots during palm's life, depending on the cultivar and In addition, offshoots are difficult to root and the success in the field transfer is usually less than 60%. One more disadvantage of this method is the spread of dangerous diseases and pests such as Bayoud disease or Red Palm Weevil which can be transported by contaminated offshoots.

13 3. In Vitro Propagation: Since conventional propagation techniques are limited in terms of providing insufficient numbers of date palm plants, biotechnology has provided a promising alternative to meet the increasing demand for healthy date palm vitro-plants Plant tissue culture techniques have been used to clone large numbers of disease-free plants from selected genotypes : it is a cloning technique which involves the use of meristematic tissues isolated under sterile condition from a known healthy and superior quality female or male plant to produce large numbers of true to type plantlets in confined controlled area. The use of high quality and uniform planting material can be multiplied on a yearround basis under disease free conditions anywhere irrespective of the season and weather. Using these techniques, date palm can be micro propagated either by somatic embryogenesis in which embryos are produced from embryogenic callus and then germinated to form complete plantlets, or through organogenesis in which plantlets are produced from multiplied buds without passing through the callus stage.

14 EMBRYO VS ORGANO In this pathway, cells or callus cultures on solid media or in suspension cultures on liquid media form embryo-like structures called somatic embryos, which on germination media, produce complete plants. The primary somatic embryos are also capable of producing more embryos through secondary somatic embryogenesis. Somatic embryogenesis method is characterized by giving in vitro plants in a comparatively, shorter time as well as high propagation ranges. But the most disadvantage of this method is the possibility of mutation and abnormalities occurrence during growing in vitro which appear in the field later on (several years after, in production fields).

15 Direct Organogenesis: The organogenesis technique, based on the exploitation of meristematic tissue potentialities to form new shoots, avoids callus formation and does not use 2,4-D. The growth regulators incorporated in the media are used at the lowest possible concentration. The organogenesis technique consists of four steps: initiation of vegetative buds; bud multiplication; shoot elongation; and rooting. The success of this technique is highly dependent on the success of the first step (initiation) which requires a welltrained staff. Furthermore, most of problems encountered in the succeeding stages (multiplication, elongation, rooting) may have their origin at the initiation phase Furthermore, since shoots are directly initiated from mother tissue without passing through a callus phase, the plantlets produced are supposed to be true-to-type In this respect, UAEU laboratory choose to develop its expertise with the micro propagation via organogenesis to guaranty in vitro plantlets highly identical in their genetic and vegetative characteristics with the mother plant.

16 The application of tissue culture techniques for date palm micro propagation has many advantages, particularly: 1. Large-scale multiplication of commercial cultivars; 2. Propagation of elite and select cultivars with desirable characters (bayoud resistance, males with superior metaxenia characteristics, high yielding, etc.); 3. Production of homogeneous, vigorous and disease-free plants; 4. No seasonal effect on plant production; 5. Enables exchange of plant material without any risk of the spread of diseases or pests.

17 Organogenesis Protocols The organogenesis technic consists of servals steps:

18 Selection and Removal of Offshoots Collect the offshoots from the farms (choose the variety) Suitable offshoots for in vitro culture may have an average weight of kg, 3 5 years old, cm high or bearing 8 12 leaves must be disease free and selected from a well-known elite adult date palm tree. Once removed, the offshoot should be cleaned of soil then all roots and leaves severely cut back before transportation to the lab. The best time period for starting in vitro culture from offshoots is between the end of date fruit harvest and the start of the next flowering stage.

19 Offshoot Preparation Dissection the offshoot out of the laboratory Offshoot preparation can be done with a sharp knife by removing, gradually (one by one), outer leaves and fibrous tissues at their bases until exposure of the shoot tip zone The ultimate size of the excised shoot tip should be about 3 4 cm in width and 6 8 cm in length.

20 Offshoots pre decontamination Once removed, the shoot tip should be transferred to an antioxidant solution containing 100 mg ascorbic acid and 150 mg citric acid to avoid tissue browning due to the phenolic compounds.

21 Shoot Tip Disinfection Disinfection procedures under laminar flow hood The excised shoot tips can be disinfected according to the following steps: (1) clean the shoot tips with distilled water to remove any organic debris

22 Disinfection procedures under laminar flow (2) soak in a fungicide solution (benomyl, mancozeb) for min; (3) rinse three times with sterile distilled water; (4) soak again, for 20 min, in a commercial Clorox solution (sodium hypochlorite) supplemented with 0.3 g/l potassium permanganate; (5) rinse three times with sterile distilled water under aseptic conditions

23 Rinsing with distilled sterilized water

24 Necessary materials

25 removing all external leaves and woody parts Explant Removal After sterilization, the root tip can be dissected to extract cultured explants. Using scalpel and forceps, the young leaves surrounding the apical dome are gradually removed.

26 Meristems + Axillary buds

27 Shoot tips (5 mm length) and axillary buds (3-5 mm length) from single nodal segments were collected and cultures in the test tubes the entire shoot tip can be cut into 4 6 pieces and transferred to a culture medium

28 Initiation phase After transfer into culture media, explants are incubated for 8 24 months in the dark so as to enhance bud initiation and also to prevent oxidation of phenolic compounds which occurs under light conditions Explants should be transferred to fresh media each month. After initiation

29 Development explants after one to two years of initiation stage

30 First reaction 14 to 16 months Shoot Formation Depending on the genotype, shoot formation generally requires months

31 Buds development 18 to 20 months Bud initiation is controlled by several factors that may act in concert Those factors in particular are the culture media components, genotype and time period of plant material collection Concerning the time period of plant material collection, it was shown that the best period coincides with the dormancy stage in advance of flowering

32 Multiplication stage (10) cycles month Once initiated, vegetative buds should be transferred gradually to lighted conditions with a photoperiod of 16 h.

33 Induction Stage

34 Elongation Stage

35 Elongation Stage

36 Rooting stage

37 Rooting stage

38 Acclimatization phase Accordingly, the acclimatization phase is the most important stage in the protocol of date palm micropropagation, because if not optimized, the whole process will be inefficient. Hence, it is not acceptable to produce a huge number of plantletsand lose them at this final stage. In fact, major differences exist between the environment of plants growing in tissue culture and those in the greenhouse; particularly, differences in light, both quantity and quality, relative humidity, nutrients and other growth promoters, the gaseous composition and the medium substrate

39 Acclimatization stage

40 Acclimatization stage

41 Acclimatization stage

42 Hardening Stage

43 Vitroplants ready to be cultivated in the farms

44 Date palm tissue culture plantation production phase