In this article we will discuss about the Culture of Shoot Apex and Leaf.
Culture of Shoot Apex:
White (’33) first attempted to culture the shoot apex of Stellaria media, but he was unsuccessful. Loo (’45) successfully cultured 5 mm long excised shoot tips of Asparagus officinalis. Cultured shoots produce cladophylls, which on subculture produce plantlets.
On agar medium containing inorganic salts and glucose Morel cultured the shoot tip of orchid Cymbidium, which produces callus (Fig. 8). This callus produces proto-corms at certain regions. Proto-corm formation increases when the shoot tips ire cultured on agitated liquid medium. Proto-corm on sub-culturing produce plantlets.
Shoot tips were successfully cultured in many other plants, such as, carnation, cauliflower and tobacco etc. Isolated shoot tips of ferns produce plantlets on a simple medium. But shoot tips of angiosperms require a complicated medium.
The size of the shoot tip selected for culture is important. According to Ball (’46) root initiation and plantlet formation can take place in culture of shoot tip containing at least three leaf primordia. But Smith and Murashige (’70) was able to get plantlets from explants containing only meristematic dome, without any leaf primordia in Daucus carota, Coleus blumei, Nicotiana sp. and Tropeolum majus etc.
Cutter (’65) distinguished apical meristem culture and shoot apex culture. Apical meristem includes apex distal to the young leaf primordia. Shoot apex includes apex and a few adjacent leaf primordia.
In orchids sometimes 5-10 mm long shoot apex is used for culture. Smith and Murashige (’70) used 80 µm long apical meristem for culture, but such small explants are very difficult to survive in culture. Larger explants have greater chance of survival and they grow more rapidly.
Shoot apex culture of woody plants have some problems. Buds from young growing shoots can be easily cultured, but buds from the mature shoots are difficult to culture. Buds from woody plants need an exogenous supply of cytokinin and gibberellin for growth.
The explants are transferred to a medium without growth hormone. This favours shoot elongation. Then the explants are transferred to a medium supplemented with exogenous auxin, which helps root initiation. Tissues with high phenolic content are difficult to culture. Due to injury polyphenol is oxidised to a dark brown coloured growth inhibiting substance. This has been observed in Eucalyptus grandis.
This difficulty can be removed by:
(a) Adding antioxidants to the medium,
(b) Pre-soaking the explants in antioxidants,
(c) Initial culture is grown in absence of light and
(d) Immediate sub-culturing on a fresh medium when the tissue appears brown.
In Eucalyptus the explants are soaked in double distilled water for three hours. This removes & phenolic compounds. The explants are then incubated in darkness for several days.
Murashige and Skoog’s medium is usually used for shoot tip culture. It is supplemented with vitamins, sucrose and suitable hormones. The culture is grown either on solid agar medium or on a filter paper bridge over a liquid medium.
In some plants the medium is supplemented with thiamine. Presence of napthalacetic acid (NAA) is required for root initiation in certain plants, such as, carnation. After root initiation the shoot is placed on a medium without IAA. Within 3-4 weeks roots develop and small leaves are formed. The plantlets produced can be transferred to soil.
Addition of malachite green or 2-4-D or thiouracil helps the growth of shoot tips. For chlorophyll formation 1000 lux fluorescent lamp is used during initial stages s and 3,000—10,000 lux lamp is used in later stages. The photoperiod is 16 hours.
During initial stages cytokinin may be added in the form of kinetin or zeatin or benzyladenine. Auxin may be used as IAA, IBA (indole 3-butyric acid) or NAA. Gibberellin is required for certain plants, such as, Chrysenthemum, Dianthtis, Solatium tuberosum etc.
During later stages IBA or NAA may be added in low concentration for a short time. This helps root initiation. Long period of auxin treatment may result in callus formation. In some plants root initiation can take place in auxin free medium.
From healthy and actively growing shoot tip explant is selected for culture. In various plants, such as carnation, chrysenthemum etc. usually a larger portion of the shoot is cut from the plant and later the older leaves are removed with a sterile scalpel (Fig. 9). The dome-shaped apical meristem with 1 or 2 leaf primordia is suitable for culture.
Usually ten mm long shoot tips are removed from healthy plants and surface sterilised in hypochlorite solution for about 15 minutes. Explants are washed in double distilled water several times and placed on a petridish lined with filter paper for dissecting the shoot apex.
The filter paper is moistened with double distilled water. Terminal 0.5 mm of the shoot apex is carefully excised and is placed in the depression (position V) of the filter paper bridge, which is folded in the form of letter M (Fig. 10).
Shoot tips of various sizes may be cut and tested for optimal size of the explant for culture. The culture tube contains about 3 c.c. Of Murashige and Skoog’s medium.
The arms of the folded filter paper is dipped in the liquid medium. The tubes are plugged with non-absorbent cotton and aluminium foil and placed in a plant growth chamber at 27°G at an intensity of 1000 lux and a photoperiod of 16 hours at 70% relative humidity.
Depending upon the material lateral bud formation requires few weeks to several month time. Potato shoot tip grows quickly and form several buds within 5-6 weeks. For root initiation these are transferred to a medium lacking BAP. After rooting plantlets are transferred to sterile soil mixture in a humid condition. The plantlets are transferred first to a greenhouse and finally to a field.
Murashige (’74) noted that in a shoot tip culture bud formation is induced by cytokinin. After the formation of several buds, each bud is separated and transferred to a fresh medium. In this way many millions plantlets can be produced from a single shoot apex within a year.
(1) By apical meristem culture pathogen-free clones can be produced very rapidly.
(2) Morel (’60) first recognised the importance of shoot tip culture for multiplication of clones.
It is a very important method of vegetative propagation and used in orchids, where vegetative propagation is very slow.
Culture of Leaf:
Sussex and coworkers successfully cultured very small (about 1.2 mm) excised leaf primordia from underground buds of Osmunda in simple culture medium containing mineral salts and sucrose. The leaves produced on such a culture are smaller than normal.
Steaves working with the fern Osmunda cinnamomea noted that very small leaf primordia (measuring about 300 µm or larger) on culture can produce shoots (Fig. 11). But larger the leaf primordia at excision there is a greater chance of leaf formation. Leaf primordia of 800 µm on culture always produce leaves. He also cultured young leaf primordia of Nicotiana and Helianthus.
Excised leaves are surface sterilized and then cultured on solidified agar medium. Such cultures can be maintained for a long period. Growth of the leaves on the culture medium depends upon the stage of the leaves during excision. Explants from young leaves grow better than older leaves.